Discussion post 250 words and two scholarly sources

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After studying this chapter you should be able to: Explain how alcohol is absorbed into the bloodstream, transported throughout the body, and eliminated by oxidation and excretion

Understand the process by which alcohol is excreted in the breath via the lungs

Understand the concepts of infrared and fuel cell breath-testing devices for alcohol testing

Describe commonly employed field sobriety tests to assess alcohol impairment

List and contrast laboratory procedures for measuring the concentration of alcohol in the blood

Relate the precautions to be taken to properly preserve blood in order to analyze its alcohol content

Understand the significance of implied-consent laws and the Schmerber v. California and Missouri v. McNeely cases to traf- fic enforcement

Describe techniques that forensic toxicologists use to isolate and identify drugs and poisons

Appreciate the significance of finding a drug in human tissues and organs to assessing impairment

Understand the drug recognition expert program and how to coordinate it with a forensic toxicology result

forensic toxicology

absorption acid alveoli anticoagulant artery base capillary excretion fuel cell detector metabolism oxidation pH scale preservative toxicologist vein

KEY TERMS

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It is no secret that in spite of the concerted efforts of law enforcement agencies to prevent distri- bution and sale of illicit drugs, thousands die every year from intentional or unintentional admin- istration of drugs, and many more innocent lives are lost as a result of the erratic and frequently uncontrollable behavior of individuals under the influence of drugs. But one should not automati- cally attribute these occurrences to the wide proliferation of illicit-drug markets. For example, in the United States alone, drug manufacturers produce enough sedatives and antidepressants each year to provide every man, woman, and child with about 40 pills. All of the statistical and medi- cal evidence shows ethyl alcohol, a legal over-the-counter drug, to be the most heavily abused drug in Western countries.

Role of Forensic Toxicology Because the uncontrolled use of drugs has become a worldwide problem affecting all segments of society, the role of the toxicologist has taken on new and added significance. Toxicologists detect and identify drugs and poisons in body fluids, tissues, and organs. Their services are required not only in such legal institutions as crime laboratories and medical examiners’ offices; they also reach into hospital laboratories—where the possibility of identifying a drug overdose may represent the difference between life and death—and into various health facilities responsible for monitoring the intake of drugs and other toxic substances. Primary examples include perform- ing blood tests on children exposed to leaded paints or analyzing the urine of addicts enrolled in methadone maintenance programs.

The role of the forensic toxicologist is limited to matters that pertain to violations of crimi- nal law. However, the responsibility for performing toxicological services in a criminal justice system varies considerably throughout the United States. In systems with a crime laboratory independent of the medical examiner, this responsibility may reside with one or the other or may be shared by both. Some systems, however, take advantage of the expertise residing in gov- ernmental health department laboratories and assign this role to them. Nevertheless, whatever facility handles this work, its caseload will reflect the prevailing popularity of the drugs that are abused in the community. In most cases, this means that the forensic toxicologist handles numer- ous requests relating to the determination of the presence of alcohol in the body.

All of the statistical and medical evidence shows that ethyl alcohol—a legal, over-the-counter substance—is the most heavily abused drug in Western countries. Forty percent of all traffic deaths in the United States, nearly 17,500 fatalities per year, are alcohol related, along with more than 2 million injuries each year requiring hospital treatment. This highway death toll, as well as the untold damage to life, limb, and property, shows the dangerous consequences of alcohol abuse. Because of the prevalence of alcohol in the toxicologist’s work, we will begin by taking a closer look at how the body processes and responds to alcohol.

Toxicology of Alcohol The subject of alcohol analysis immediately confronts us with the primary objective of forensic toxicology: to detect and isolate drugs in the body so that their influence on human behavior can be determined. Knowing how the body metabolizes alcohol provides the key to understanding its effects on human behavior. This knowledge has also made possible the development of instru- ments that measure the presence and concentration of alcohol in individuals suspected of driving while under its influence.

Metabolism of Alcohol All chemicals that enter the body are eventually broken down by chemicals within the body and transformed into other chemicals that are easier to eliminate. This process of transformation, called metabolism, consists of three basic steps: absorption, distribution, and elimination.

ABSORPTION AND DISTRIBUTION Alcohol, or ethyl alcohol, is a colorless liquid normally diluted with water and consumed as a beverage. Alcohol appears in the blood within minutes after it has been consumed and slowly increases in concentration while it is being absorbed

metabolism The transformation of a chemical in the body to another chemical to facilitate its elimination from the body.

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from the stomach and the small intestine into the bloodstream. During the absorption phase, alcohol slowly enters the body’s bloodstream and is carried to all parts of the body. When the absorption period is completed, the alcohol becomes distributed uniformly throughout the watery portions of the body—that is, throughout about two-thirds of the body volume. Fat, bones, and hair are low in water content and therefore contain little alcohol, whereas alcohol concentration in the rest of the body is fairly uniform. After absorption is completed, a maximum alcohol level is reached in the blood, and the postabsorption period begins. Then the alcohol concentration slowly decreases until it reaches zero again.

Many factors determine the rate at which alcohol is absorbed into the bloodstream, includ- ing the total time taken to consume the drink, the alcohol content of the beverage, the amount consumed, and the quantity and type of food present in the stomach at the time of drinking. With so many variables, it is difficult to predict just how long the absorption process will require. For example, beer is absorbed more slowly than an equivalent concentration of alcohol in water, apparently because of the carbohydrates in beer. Also, alcohol consumed on an empty stomach is absorbed faster than an equivalent amount of alcohol taken when there is food in the stomach (see Figure 12–1).

The longer the total time required for complete absorption to occur, the lower the peak alcohol concentration in the blood. Depending on a combination of factors, maximum blood-alcohol concentration may not be reached until two or three hours have elapsed from the time of consumption. However, under normal social drinking conditions, it takes any- where from 30 to 90 minutes from the time of the final drink until the absorption process is completed.

ELIMINATION As the alcohol is circulated by the bloodstream, the body begins to eliminate it. Alcohol is eliminated through two mechanisms: oxidation and excretion. Nearly all of the alcohol consumed (95 to 98 percent) is eventually oxidized to carbon dioxide and water. Oxidation takes place almost entirely in the liver. There, in the presence of the enzyme alcohol dehydrogenase, the alcohol is converted into acetaldehyde and then to acetic acid. The acetic acid is subsequently oxidized in practically all parts of the body, becoming carbon dioxide and water.

The remaining alcohol is excreted, unchanged, in the breath, urine, and perspiration. Most significant, the amount of alcohol exhaled in the breath is in direct proportion to the concentra- tion of alcohol in the blood. This observation has had a tremendous impact on the technology and

absorption Passage of alcohol across the wall of the stomach and small intestine into the bloodstream.

oxidation The combination of oxygen with other substances to produce new products.

excretion Elimination of alcohol from the body in an unchanged state; alcohol is normally excreted in breath and urine.

FIGURE 12–1 Blood-alcohol concentrations after ingestion of 2 ounces of pure alcohol mixed in 8 ounces of water (equivalent to about 5 ounces of 80-proof vodka). Source: Courtesy U.S. Department of Transportation, Washington, D.C.

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Pulmonary artery

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RA LA

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Simplified diagram of the human circulatory system. Dark vessels contain oxygenated blood; light vessels contain deoxygenated blood.

Alcohol in the Circulatory System

The extent to which an individual may be under the influence of alcohol is usually determined by mea- suring the quantity of alcohol present in the blood system. Normally, this is accomplished in one of two ways: (1) by direct chemical analysis of the blood for its alcohol content or (2) by measurement of the al- cohol content of the breath. In either case, the sig- nificance and meaning of the results can better be understood when the movement of alcohol through the circulatory system is studied.

Humans, like all vertebrates, have a closed circu- latory system, which consists basically of a heart and numerous arteries, capillaries, and veins. An artery is a blood vessel carrying blood away from the heart, and a vein is a vessel carrying blood back toward the heart. Capillaries are tiny blood vessels that interconnect the arteries with the veins. The exchange of materials between the blood and the other tissues takes place across the thin walls of the capillaries. A schematic dia- gram of the circulatory system is shown in the figure.

Ingestion and Absorption Let us now trace the movement of alcohol through the human circulatory system. After alcohol is ingested, it

inside the science moves down the esophagus into the stomach. About 20 percent of the alcohol is absorbed through the stom- ach walls into the portal vein of the blood system. The remaining alcohol passes into the blood through the walls of the small intestine. Once in the blood, the alco- hol is carried to the liver, where its destruction starts as the blood (carrying the alcohol) moves up to the heart.

The blood enters the upper right chamber of the heart, called the right atrium (or auricle), and is forced into the lower right chamber of the heart, known as the right ventricle. Having returned to the heart from its circulation through the tissues, the blood at this time contains very little oxygen and much carbon di- oxide. Consequently, the blood must be pumped up to the lungs, through the pulmonary artery, to be re- plenished with oxygen.

Aeration The respiratory system bridges with the circulatory sys- tem in the lungs, so that oxygen can enter the blood and carbon dioxide can leave it. As shown in the fig- ure, the pulmonary artery branches into capillaries ly- ing close to tiny pear-shaped sacs called alveoli. The lungs contain about 250 million alveoli, all located at the ends of the bronchial tubes. The bronchial tubes connect to the windpipe (trachea), which leads up to the mouth and nose (see the figure). At the surface of the alveolar sacs, blood flowing through the capillar- ies comes in contact with fresh oxygenated air in the sacs. A rapid exchange now proceeds to take place between the fresh air in the sacs and the spent air in the blood. Oxygen passes through the walls of the alveoli into the blood while carbon dioxide is dis- charged from the blood into the air (see the figure). If, during this exchange, alcohol or any other volatile substance is in the blood, it too will pass into the al- veoli. During breathing, the carbon dioxide and al- cohol are expelled through the nose and mouth, and the alveoli sacs are replenished with fresh oxygenated air breathed into the lungs, allowing the process to begin all over again.

The distribution of alcohol between the blood and alveolar air is similar to the example of a gas dis- solved in an enclosed beaker of water, as described on page 280. Here again, one can use Henry’s law to explain how the alcohol divides itself between the air and blood. Henry’s law may now be restated as follows: When a volatile chemical (alcohol) is dis- solved in a liquid (blood) and is brought to equi- librium with air (alveolar breath), there is a fixed ratio between the concentration of the volatile compound (alcohol) in air (alveolar breath) and its concentration in the liquid (blood), and this ratio is constant for a given temperature.

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tiny veins that fuse to form larger veins. These veins eventually lead back to the heart to complete the circuit.

During absorption, the concentra- tion of alcohol in the arterial blood is considerably higher than the concentra- tion of alcohol in the venous blood. One typical study revealed a subject’s arte- rial blood-alcohol level to be 41 percent higher than the venous level 30 minutes after the last drink.1 This difference is thought to exist because of the rapid diffusion of alcohol into the body tis- sues from venous blood during the early phases of absorption. Because the administration of a blood test requires drawing venous blood from the arm, this test is clearly to the advantage of a sub- ject who may still be in the absorption stage. However, once absorption is com- plete, the alcohol becomes equally dis- tributed throughout the blood system.

The temperature at which the breath leaves the mouth is normally 34°C. At this temperature, experimental evidence has shown that the ra- tio of alcohol in the blood to alcohol in alveoli air is approximately 2,100 to 1. In other words, 1 milliliter of blood will contain nearly the same amount of alcohol as 2,100 milliliters of alveo- lar breath. Henry’s law thus becomes a basis for relating breath to blood-alcohol concentration.

Recirculation and Distribution Now let’s return to the circulating blood. After emerging from the lungs, the oxygenated blood is rushed back to the upper left chamber of the heart (left atrium) by the pulmonary vein. When the left atrium contracts, it forces the blood through a valve into the left ventricle, which is the lower left cham- ber of the heart. The left ventricle then pumps the freshly oxygenated blood into the arteries, which carry the blood to all parts of the body. Each of these arteries, in turn, branches into smaller arter- ies, which eventually connect with the numerous tiny capillaries embedded in the tissues. Here the alcohol moves out of the blood and into the tis- sues. The blood then runs from the capillaries into

Pulmonary artery

Pulmonary vein

Bronchial tube

Carbon dioxide Alveolar sac

Carbon dioxide

Oxygen Alveolar sac

Oxygen

Gas exchange in the lungs. Blood flows from the pulmonary artery into vessels that lie close to the walls of the alveoli sacs. Here the blood gives up its carbon dioxide and absorbs oxygen. The oxygenated blood leaves the lungs via the pulmonary vein and returns to the heart.

Nasal cavity

Larynx

Trachea Esophagus

Bronchial tube

Alveolar sac

The respiratory system. The trachea connects the nose and mouth to the bronchial tubes. The bronchial tubes divide into numerous branches that terminate in the alveoli sacs in the lungs.

1 R. B. Forney et al., “Alcohol Distribution in the Vascular System: Concen- trations of Orally Administered Alcohol in Blood from Various Points in the Vascular System and in Rebreathed Air during Absorption,” Quarterly Journal of Studies on Alcohol 25 (1964): 205.

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artery A blood vessel that carries blood away from the heart.

vein A blood vessel that transports blood toward the heart.

capillary A tiny blood vessel across whose walls exchange of materials be- tween the blood and the tissues takes place; it receives blood from arteries and carries it to veins.

alveoli Small sacs in the lungs through whose walls air and other vapors are exchanged between the breath and the blood.

procedures used for blood-alcohol testing. The development of instruments to reliably measure breath for its alcohol content has made possible the testing of millions of people in a quick, safe, and convenient manner.

The fate of alcohol in the body is therefore relatively simple—namely, absorption into the bloodstream, distribution throughout the body’s water, and finally, elimination by oxida- tion and excretion. The elimination, or “burn-off,” rate of alcohol varies in different individ- uals; 0.015 percent w/v (weight per volume) per hour is the average rate after the absorption process is complete.2 However, this figure is an average that varies by as much as 30 percent among individuals.

BLOOD-ALCOHOL CONCENTRATION Logically, the most obvious measure of intoxication would be the amount of liquor a person has consumed. Unfortunately, most arrests are made after the fact, when such information is not available to legal authorities; furthermore, even if these data could be collected, numerous related factors, such as body weight and the rate of alcohol’s absorption into the body, are so variable that it would be impossible to prescribe uniform standards that would yield reliable alcohol intoxication levels for all individuals.

Theoretically, for a true determination of the quantity of alcohol impairing an individual’s normal body functions, it would be best to remove a portion of brain tissue and analyze it for alcohol content. For obvious reasons, this cannot be done on living subjects. Consequently, toxicologists concentrate on the blood, which provides the medium for circulating alcohol throughout the body, carrying it to all tissues including the brain. Fortunately, experimental evidence supports this approach and shows blood-alcohol concentration to be directly pro- portional to the concentration of alcohol in the brain. From the medicolegal point of view, blood-alcohol levels have become the accepted standard for relating alcohol intake to its effect on the body.

As noted earlier, alcohol becomes concentrated evenly throughout the watery portions of the body. This knowledge can be useful for the toxicologist analyzing a body for the presence of al- cohol. If blood is not available, as in some postmortem situations, a medical examiner can select a water-rich organ or fluid—for example, the brain, cerebrospinal fluid, or vitreous humor—to estimate the body’s equivalent alcohol level.

Testing for Intoxication From a practical point of view, drawing blood from veins of motorists suspected of being under the influence of alcohol is simply not convenient. The need to transport each suspect to a loca- tion where a medically qualified person can draw blood would be costly and time consuming, considering the hundreds of suspects that the average police department must test every year. The methods used must be designed to test hundreds of thousands of motorists annually, without causing them undue physical harm or unreasonable inconvenience, and provide a reliable diagno- sis that can be supported and defended within the framework of the legal system. This means that toxicologists have had to devise rapid and specific procedures for measuring a driver’s degree of alcohol intoxication that can be easily administered in the field.

Breath Testing for Alcohol The most widespread method for rapidly determining alcohol intoxication is breath testing. A breath tester is simply a device for collecting and measuring the alcohol content of alveolar breath. Alcohol is expelled, unchanged, in the breath of a person who has been drinking. A breath test measures the alcohol concentration in the pulmonary artery by measuring its concentration in alveolar breath. Thus, breath analysis provides an easily obtainable specimen along with a rapid and accurate result.

2 In the United States, laws that define blood-alcohol levels almost exclusively use the unit percent weight per volume—% w/v. Hence, 0.015 percent w/v is equivalent to 0.015 gram of alcohol per 100 milliliters of blood, or 15 milligrams of alcohol per 100 milliliters.

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Breath-test results obtained during the absorption phase may be higher than results obtained from a simultaneous analysis of venous blood. However, the former are more reflective of the concentration of alcohol reaching the brain and therefore more accurately reflect the effects of alcohol on the subject. Again, once absorption is complete, the difference between a blood test and a breath test should be minimal.

BREATH-TEST INSTRUMENTS The first widely used instrument for measuring the alcohol content of alveolar breath was the Breathalyzer, developed in 1954 by R. F. Borkenstein, who was a captain in the Indiana State Police. Starting in the 1970s, the Breathalyzer was phased out and replaced by other instruments. Like the Breathalyzer, they assume that the ratio of alcohol in the blood to alcohol in alveolar breath is 2,100 to 1 at a mouth temperature of 34°C. In other words, 1 milliliter of blood contains nearly the same amount of alcohol as 2,100 milliliters of alveolar breath. Unlike the Breathalyzer, modern breath testers are free of chemicals. These devices include infrared light–absorption devices and fuel cell detectors (described in the following “Inside the Science” box).

Infrared and fuel-cell-based breath testers are microprocessor controlled, so all an operator has to do is to press a start button; the instrument automatically moves through a sequence of steps and produces a readout of the subject’s test results. These instru- ments also perform self-diagnostic tests to ascertain whether they are in proper operating condition.

CONSIDERATIONS IN BREATH TESTING An important feature of these instruments is that they can be connected to an external alcohol standard or simulator in the form of either a liquid or a gas. The liquid simulator contains a known concentration of alcohol in water. It is heated to a controlled temperature and the vapor formed above the liquid is pumped into the instrument. Dry-gas standards typically consist of a known concentration of alcohol mixed with an inert gas and compressed in cylinders. The external standard is automatically sampled by the breath-test instrument before and/or after the subject’s breath sample is taken and recorded. Thus the operator can check the accuracy of the instrument against the known alcohol standard.

The key to the accuracy of a breath-testing device is to ensure that the unit captures the alcohol in the alveolar (i.e., deep-lung) breath of the subject. This is typically accomplished by programming the unit to accept no less than 1.1 to 1.5 liters of breath from the subject. Also, the subject must blow for a minimum time (such as 6 seconds) with a minimum breath flow rate (such as 3 liters per minute).

The breath-test instruments just described feature a slope detector, which ensures that the breath sample is alveolar, or deep-lung, breath. As the subject blows into the instrument, the breath-alcohol concentration is continuously monitored. The instrument accepts a breath sample only when consecutive measurements fall within a predetermined rate of change. This approach ensures that the sample measurement is deep-lung breath and closely relates to the true blood- alcohol concentration of the subject being tested.

A breath-test operator must take other steps to ensure that the breath-test result truly reflects the actual blood-alcohol concentration within the subject. A major consideration is to avoid mea- suring “mouth alcohol” resulting from regurgitation, belching, or recent intake of an alcoholic beverage. Also, recent gargling with an alcohol-containing mouthwash can lead to the presence of mouth alcohol. As a result, the alcohol concentration detected in the exhaled breath is higher than the concentration in the alveolar breath. To avoid this possibility, the operator must not al- low the subject to take any foreign material into his or her mouth for at least fifteen minutes be- fore the breath test. Likewise, the subject should be observed not to have belched or regurgitated during this period. Mouth alcohol has been shown to dissipate after fifteen to twenty minutes from its inception.

Measurement of independent breath samples taken within a few minutes of each other is another extremely important check of the integrity of the breath test. Acceptable agree- ment between the two tests taken minutes apart significantly reduces the possibility of er- rors caused by the operator, mouth alcohol, instrument component failures, and spurious electric signals.

fuel cell detector A detector in which chemical reac- tions produce electricity.

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306 CHAPTER 12

Infrared Light Absorption

In principle, infrared instruments operate no dif- ferently than the spectrophotometers described in Chapter 11. An evidential testing instrument that incorporates the principle of infrared light ab- sorption is shown in Figure 1. Any alcohol present in the subject’s breath flows into the instrument’s breath chamber. As shown in Figure 2, a beam of infrared light is aimed through the chamber. A fil- ter is used to select a wavelength of infrared light at which alcohol will absorb. As the infrared light passes through the chamber, it interacts with the alcohol and causes the light to decrease in inten- sity. The decrease in light intensity is measured by a photoelectric detector that gives a signal proportional to the concentration of alcohol pres- ent in the breath sample. This information is pro- cessed by an electronic microprocessor, and the percent blood-alcohol concentration is displayed on a digital readout. Also, the blood-alcohol level is printed on a card to produce a permanent rec- ord of the test result. Most infrared breath testers aim a second infrared beam into the same cham- ber to check for acetone or other chemical inter- ferences on the breath. If the instrument detects differences in the relative response of the two infrared beams that does not conform to ethyl alcohol, the operator is immediately informed of the presence of an “interferant.”

inside the science

DetectorInfrared radiation source

Sample chamber Filter

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Breath outlet

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Infrared light beamed through chamber. Alcohol in breath absorbs some infrared light.

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(a) An infrared breath-testing instrument—the Data Master DMT. (b) A subject blowing into the DMT breath tester.

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Field Sobriety Testing A police officer who suspects that an individual is under the influence of alcohol usually conducts a series of preliminary tests before ordering the suspect to submit to an evidential breath or blood test. These preliminary, or field sobriety, tests are normally performed to ascertain the degree of the suspect’s physical impairment and whether an evidential test is justified.

Field sobriety tests usually consist of a series of psychophysical tests and a preliminary breath test (if such devices are authorized and available for use). A portable handheld roadside breath tester is shown in Figure 12–2. This pocket-sized device weighs 5 ounces and uses a fuel cell to measure the alcohol content of a breath sample. The fuel cell absorbs the alcohol from the breath sample, oxidizes it, and produces an electrical current proportional to the breath-alcohol content. This instrument Figure 12–2 can typically perform for years before the fuel cell needs to be replaced. Its been approved for use as an evidential breath tester by the National Highway Traffic Safety Administration.

Horizontal-gaze nystagmus, walk and turn, and the one-leg stand constitute a series of reliable and effective psychophysical tests. Horizontal-gaze nystagmus is an involuntary jerking of the eye as it moves to the side. A person experiencing nystagmus is usually unaware

Infrared radiation source

Sample chamber Filter selects wavelength of IR light at which alcohol absorbs

Breath inlet

Breath outlet

Detector

Infrared radiation source

Sample chamber

Breath inlet

Breath outlet

Detector converts infrared light to an electrical signal proportional to the alcohol content in breath.

Infrared radiation source

Sample chamber

Breath inlet

Breath outlet

Detector Breath-alcohol content is converted into a blood-alcohol concentration and displayed on a digital readout.

Schematic diagram of an infrared breath-testing instrument.

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FIGURE 12–2 (a) The Alco-Sensor FST. (b) A subject blowing into the roadside tester device.

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The Fuel Cell

A fuel cell converts energy arising from a chemical re- action into electrochemical energy. A typical fuel cell consists of two platinum electrodes separated by an acid- or base-containing porous membrane. A plati- num wire connects the electrodes and allows a current to flow between them. In the alcohol fuel cell, one of the electrodes is positioned to come into contact with a subject’s breath sample. If alcohol is present in the breath, a reaction at the electrode’s surface converts the alcohol to acetic acid. One by-product of this con- version is free electrons, which flow through the con- necting wire to the opposite electrode, where they interact with atmospheric oxygen to form water (see the figure). The fuel cell also requires the migration of hydrogen ions across the acidic porous membrane to complete the circuit. The strength of the current flow between the two electrodes is proportional to the concentration of alcohol in the breath.

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A fuel cell detector in which chemical reactions are used to produce electricity.

that the jerking is happening and is unable to stop or control it. The subject being tested is asked to follow a penlight or some other object with his or her eye as far to the side as the eye can go. The more intoxicated the person is, the less the eye has to move toward the side before jerking or nystagmus begins. Usually, when a person’s blood-alcohol concentration is in the range of 0.10 percent, the jerking begins before the eyeball has moved 45 degrees to the side

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(see Figure 12–3). Higher blood-alcohol concentration causes jerking at smaller angles. Also, if the suspect has taken a drug that also causes nystagmus (such as phencyclidine, barbiturates, and other depressants), the nystagmus onset angle may occur much earlier than would be expected from alcohol alone.

Walk and turn and the one-leg stand are divided-attention tasks, testing the subject’s abil- ity to comprehend and execute two or more simple instructions at one time. The ability to understand and simultaneously carry out more than two instructions is significantly affected by increasing blood-alcohol levels. Walk and turn requires the suspect to maintain balance while standing heel-to-toe and at the same time listening to and comprehending the test instructions. During the walking stage, the suspect must walk a straight line, touching heel-to-toe for nine steps, then turn around on the line and repeat the process. The one-leg stand requires the sus- pect to maintain balance while standing with heels together listening to the instructions. Dur- ing the balancing stage, the suspect must stand on one foot while holding the other foot several inches off the ground for 30 seconds; simultaneously, the suspect must count out loud during the 30-second time period.

Analysis of Blood for Alcohol Gas chromatography is the approach most widely used by forensic toxicologists for determining alcohol levels in blood. Under proper gas chromatographic conditions, alcohol can be separated from other volatile substances in the blood. By comparing the resultant alcohol peak area to ones obtained from known blood-alcohol standards, the investigator can calculate the alcohol level with a high degree of accuracy (see Figure 12–4).

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FIGURE 12–3 When a person’s blood-alcohol level is in the range of 0.10 percent, jerking of the eye during the horizontal-gaze nystagmus test begins before the eyeball has moved 45 degrees to the side.

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FIGURE 12–4 A gas chromatogram showing ethyl alcohol (ethanol) in whole blood.

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Another procedure for alcohol analysis involves the oxidation of alcohol to acetalde- hyde. This reaction is carried out in the presence of the enzyme alcohol dehydrogenase and the coenzyme nicotin-amide-adenine dinucleotide (NAD). As the oxidation proceeds, NAD is converted into another chemical species, NADH. The extent of this conversion is measured by a spectrophotometer and is related to alcohol concentration. This approach to blood-alcohol testing is normally associated with instruments used in clinical or hospital settings. Instead, forensic laboratories normally use gas chromatography for determining blood-alcohol content.

Collection and Preservation of Blood Blood must always be drawn under medically acceptable conditions by a qualified individual. A nonalcoholic disinfectant should be applied before the suspect’s skin is penetrated with a ster- ile needle or lancet. It is important to eliminate any possibility that an alcoholic disinfectant could inadvertently contribute to a falsely high blood-alcohol result. Nonalcoholic disinfectants such as aqueous benzalkonium chloride (Zepiran), aqueous mercuric chloride, or povidone-iodine (Betadine) are recommended for this purpose.

Once blood is removed from an individual, it is best preserved sealed in an airtight container after adding an anticoagulant and a preservative. The blood should be stored in a refrigerator until delivery to the toxicology laboratory. The addition of an anticoagulant, such as EDTA or potassium oxalate, prevents clotting; a preservative, such as sodium fluoride, inhibits the growth of microorganisms capable of destroying alcohol.

One study performed to determine the stability of alcohol in blood removed from living in- dividuals found that the most significant factors affecting alcohol’s stability in blood are storage temperature, the presence of a preservative, and the length of storage.3 Not a single blood speci- men examined showed an increase in alcohol level with time. Failure to keep the blood refriger- ated or to add sodium fluoride resulted in a substantial decline in alcohol concentration. Longer storage times also reduced blood-alcohol levels. Hence, failure to adhere to any of the proper preservation requirements for blood works to the benefit of the suspect and to the detriment of society.

The collection of postmortem blood samples for alcohol-level determinations requires added precautions. Ethyl alcohol may be generated in the body of a deceased individual as a result of bacterial action. Therefore, it is best to collect a number of blood samples from different body sites. For example, blood may be removed from the heart and from the femoral vein (in the leg) and cubital vein (in the arm). Each sample should be placed in a clean, airtight container contain- ing an anticoagulant and sodium fluoride preservative and should be refrigerated. Blood-alcohol levels can be attributed solely to alcohol consumption if they are nearly similar in all blood samples collected from the same person. As an alternative to blood collection, the collection of vitreous humor and urine is recommended. Vitreous humor and urine usually do not experience any significant postmortem ethyl alcohol production.

Alcohol and the Law Constitutionally, every state in the United States is charged with establishing and administer- ing statutes regulating the operation of motor vehicles. Although such an arrangement might encourage diverse laws defining permissible blood-alcohol levels, this has not been the case. Both the American Medical Association and the National Safety Council have exerted con- siderable influence in persuading the states to establish uniform and reasonable blood-alcohol standards.

Blood-Alcohol Laws Between 1939 and 1964, 39 states and the District of Columbia enacted legislation that followed the recommendations of the American Medical Association and the National Safety Council in specifying that a person with a blood-alcohol concentration in excess of 0.15 percent w/v

3 G. A. Brown et al., “The Stability of Ethanol in Stored Blood,” Analytica Chemica Acta 66 (1973): 271.

anticoagulant A substance that prevents coagula- tion or clotting of blood.

preservative A substance that stops the growth of microorganisms in blood.

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was to be considered under the influence of alcohol.4 However, continued experimental studies have since shown a clear correlation between drinking and driving impairment for blood-alcohol levels much below 0.15 percent w/v. As a result of these studies, in 1960 the American Medical Association and in 1965 the National Safety Council recommended lowering the presumptive level at which an individual was considered to be under the influence of alcohol to 0.10 percent w/v. In 2000, U.S. federal law established 0.08 percent as the per se blood-alcohol level, meaning that any individual meeting or exceeding this blood-alcohol level shall be deemed intoxicated. No other proof of alcohol impairment is necessary. The 0.08 percent level applies only to non- commercial drivers, as the federal government has set the maximum allowable blood-alcohol concentration for commercial truck and bus drivers at 0.04 percent.

Several Western countries have also set 0.08 percent w/v as the blood-alcohol level above which it is an offense to drive a motor vehicle. Those countries include Canada, Italy, Switzerland, and the United Kingdom. Finland, France, Germany, Ireland, Japan, the Netherlands, and Norway have a 0.05 percent limit. Australian states have adopted a 0.05 percent blood-alcohol concentra- tion level. Sweden has lowered its blood-alcohol concentration limit to 0.02 percent.

As shown in Figure 12–5, one is about four times as likely to become involved in an automo- bile accident at the 0.08 percent level as a sober individual. At the 0.15 percent level, the chances are 25 times as much for involvement in an automobile accident compared to a sober driver. The reader can estimate the relationship of blood-alcohol levels to body weight and the quantity of 80-proof liquor consumed by referring to Figure 12–6.

Constitutional Issues The Fifth Amendment to the U.S. Constitution guarantees all citizens protection against self- incrimination—that is, against being forced to make an admission that would prove one’s own guilt in a legal matter. To prevent a person’s refusal to take a test for alcohol intoxication on the constitutional grounds of self-incrimination, the National Highway Traffic Safety Administration (NHTSA) recommended an “implied consent” law. By 1973, all the states had complied with this recommendation. In accordance with this statute, operating a motor vehicle on a public highway automatically carries with it the stipulation that the driver must either submit to a test for alco- hol intoxication if requested or lose his or her license for some designated period—usually six months to one year.

4 0.15 percent w/v is equivalent to 0.15 grams of alcohol per 100 milliliters of blood, or 150 milligrams per 100 milliliters.

About 25 times as much as normal at 0.15%

.00 .04 .08 .12 .16 .20 Blood-alcohol concentration

About 4 times as much as normal at 0.08%

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FIGURE 12–5 Diagram of increased driving risk in relation to blood-alcohol concentration.

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In 1966, the Supreme Court, in Schmerber v. California,5 addressed the constitutionality of collecting a blood specimen for alcohol testing, as well as for obtaining other types of physi- cal evidence from a suspect without consent. While being treated at a Los Angeles hospital for injuries sustained in an automobile collision, Schmerber was arrested for driving under the influence of alcohol. A physician took a blood sample from Schmerber at the direction of the police, over the objection of the defendant. On appeal to the U.S. Supreme Court, the defen- dant argued that his privilege against self-incrimination had been violated by the introduction of the results of the blood test at his trial. The Court ruled against the defendant, reasoning that the Fifth Amendment only prohibits compelling a suspect to give “testimonial” evidence that may be self-incriminating; being compelled to furnish “physical” evidence, such as fin- gerprints, photographs, measurements, and blood samples, the Court ruled, was not protected by the Fifth Amendment.

The Court also addressed the question of whether Schmerber was subjected to an unreason- able search and seizure by the taking of a blood specimen without a search warrant. In the 1966 decision, the Court upheld the blood removal, reasoning that the natural body elimination of alcohol created an emergency situation allowing for a warrantless search. The Court revisited this issue once again forty-seven years after Schmerber in the case of Missouri v. McNeely.6

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How to Tell What Your Blood Alcohol Level Is after Drinking

FIGURE 12–6 To use this diagram, lay a straightedge across your weight and the number of ounces of liquor you’ve consumed on an empty or full stomach. The point where the edge hits the right-hand column is your maximum blood-alcohol level. The rate of elimination of alcohol from the bloodstream is approximately 0.015 percent per hour. Therefore, to calculate your actual blood-alcohol level, subtract 0.015 from the number in the right- hand column for each hour from the start of drinking. Source: U.S. Department of Transportation.

5 384 U.S. 757 (1966). 6 133 S. Ct. 932 (2013).

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Here, the Court addressed the issue as to whether the natural elimination of alcohol in blood categorically justifies a warrantless intrusion. The Court noted that advances in com- munication technology now allow police to obtain warrant quickly by phone, e-mail, or teleconferencing.

In those drunk-driving investigations where police officers can reasonably obtain a warrant before a blood sample can be drawn without significantly undermining the efficacy of the search, the Fourth Amendment mandates that they do so. . . . In short, while the natural dissipation of alcohol in the blood may support a finding of exigency in a specific case, as it did in Schmerber, it does not do so categorically. Whether a warrantless blood test of a drunk-driving suspect is reasonable must be determined case by case based on the totality of the circumstances.

The Role of the Toxicologist Once the forensic toxicologist ventures beyond the analysis of alcohol, he or she encounters an encyclopedic maze of drugs and poisons. Even a cursory discussion of the problems and handi- caps imposed on toxicologists is enough to develop a sense of appreciation for their accomplish- ments and ingenuity.

Challenges Facing the Toxicologist The toxicologist is presented with body fluids and/or organs and asked to examine them for the presence of drugs and poisons. If he or she is fortunate, which is not often, some clue to the type of toxic substance present may develop from the victim’s symptoms, a postmortem patho- logical examination, an examination of the victim’s personal effects, or the nearby presence of empty drug containers or household chemicals. Without such supportive information, the toxicologist must use general screening procedures with the hope of narrowing thousands of possibilities to one.

If this task does not seem monumental, consider that the toxicologist is not dealing with drugs at the concentration levels found in powders and pills. By the time a drug specimen reaches the toxicology laboratory, it has been dissipated and distributed throughout the body. Whereas the drug analyst may have gram or milligram quantities of material to work with, the toxicolo- gist must be satisfied with nanogram or at best microgram amounts, acquired only after careful extraction from body fluids and organs.

Furthermore, the body is an active chemistry laboratory, and no one can appreciate this observation more than a toxicologist. Few substances enter and completely leave the body in the same chemical state. The drug that is injected is not always the substance extracted from the body tissues. Therefore, a thorough understanding of how the body alters or metabolizes the chemical structure of a drug is essential in detecting its presence.

It would, for example, be futile and frustrating to search exhaustively for heroin in the hu- man body. This drug is almost immediately metabolized to morphine on entering the blood- stream. Even with this information, the search may still prove impossible unless the examiner also knows that only a small percentage of morphine is excreted unchanged in urine. For the most part, morphine becomes chemically bonded to body carbohydrates before elimination in urine. Thus, successful detection of morphine requires that its extraction be planned in accordance with a knowledge of its chemical fate in the body.

Another example of how one needs to know how a drug metabolizes itself in the body is exemplified by the investigation of the death of Anna Nicole Smith. In her case, the sedative chloral hydrate was a major contributor to her death, and its presence was detected by its active metabolite, trichloroethanol (see the following case files box).

Last, when and if the toxicologist has surmounted all of these obstacles and has finally de- tected, identified, and quantitated a drug or poison, he or she must assess the substance’s toxicity. Fortunately, there is published information relating to the toxic levels of most drugs; however, when such data are available, their interpretation must assume that the victim’s physiological behavior agrees with that of the subjects of previous studies. In some cases, such an assumption

WEBEXTRA 12.1 Calculate Your Blood-Alcohol Level

WEBEXTRA 12.2 See How Alcohol Affects Your Behavior

toxicologist An individual charged with the responsibility of detecting and identifying the presence of drugs and poisons in body fluids, tissues, and organs.

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may not be entirely valid without knowing the subject’s case history. No experienced toxicolo- gist would be surprised to find an individual tolerating a toxic level of a drug that would have killed most other people.

Collection and Preservation of Toxicological Evidence Toxicology is made infinitely easier once it is recognized that the toxicologist’s capabilities are directly dependent on the input received from the attending physician, medical examiner, and police investigator. It is a tribute to forensic toxicologists, who must often labor under conditions that do not afford such cooperation, that they can achieve such a high level of proficiency.

Generally, with a deceased person, the medical examiner decides what biological specimens must be shipped to the toxicology laboratory for analysis. However, a living person suspected of being under the influence of a drug presents a completely different problem, and few options are available. When possible, both blood and urine are taken from any suspected drug user. The entire urine void is collected and submitted for toxicological analysis. Preferably, two consecu- tive voids should be collected in separate specimen containers.

When a licensed physician or registered nurse is available, a sample of blood should also be collected. The amount of blood taken depends on the type of examination to be con- ducted. Comprehensive toxicological tests for drugs and poisons can conveniently be car- ried out on a minimum of 10 cc of blood. A determination solely for the presence of alcohol will require much less—approximately 5 cc of blood. However, many therapeutic drugs, such as tranquilizers and barbiturates, when taken in combination with a small, nonintoxi- cating amount of alcohol, produce behavioral patterns resembling alcohol intoxication. For this reason, the toxicologist must be given an adequate amount of blood so he or she will

Michael Jackson: The Demise of a Superstar A call to 911 had the desperate tone of urgency. The voice of a young man implored an ambulance to hurry to the home of pop star Michael Jackson. The unconscious performer was in cardiac arrest and was not responding to CPR. The 50-year-old Jackson was pronounced dead upon arrival at a regional medi- cal center. When the initial autopsy results revealed no signs of foul play, rumors immediately began to swirl around a drug-related death. News media coverage showed investiga- tors carrying bags full of drugs and syringes out of the Jackson residence. So it came as no surprise that the forensic toxicology report accompanying Jackson’s autopsy showed that the enter- tainer had died of a drug overdose.

Apparently Jackson had become accustomed to receiv- ing sedatives to help him sleep. Early on the morning of his death, his physician gave Jackson a tab of Valium. At 2 a.m., he administered the sedative lorazepam, and at 3 a.m. the physi- cian administered another sedative, midazolam. Those drugs were administered again at 5 a.m. and 7:30 a.m., but Jackson still was unable to sleep. Finally, at about 10:40 a.m., Jackson’s doctor gave him 25 milligrams of propofol, at which point

Jackson went to sleep. Propofol is a powerful sedative used primarily in the maintenance of surgical anesthesia. All of the drugs administered to Jackson were sedatives, which can act in concert to depress the activities of the central nervous system. Therefore, it comes as no surprise that this drug cocktail re- sulted in cardiac arrest and death.

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have the option of performing a comprehensive analysis for drugs in cases of low alcohol concentrations.

Techniques Used in Toxicology For the toxicologist, the upsurge in drug use and abuse has meant that the overwhelming major- ity of fatal and nonfatal toxic agents are drugs. Not surprisingly, a relatively small number of drugs—namely, those discussed in Chapter 11—comprise nearly all the toxic agents encoun- tered. Of these, alcohol, marijuana, and cocaine normally account for 90 percent or more of the drugs encountered in a typical toxicology laboratory.

ACIDS AND BASES Like the drug analyst, the toxicologist must devise an analytical scheme to detect, isolate, and identify a toxic substance. The first chore is to selectively remove and isolate drugs and other toxic agents from the biological materials submitted as evidence. Because drugs constitute a large portion of the toxic materials found, a good deal of effort must be devoted to their extraction and detection. The procedures are numerous, and a useful description of them would be too detailed for this text. We can best understand the underlying principle of drug extraction by observing that many drugs fall into the categories of acids and bases.

Although several definitions exist for these two classes, a simple one states that an acid is a compound that sheds a hydrogen ion (or a hydrogen atom minus its electron) with reasonable

Accidental Overdose: The Tragedy of Anna Nicole Smith Rumors exploded in the media when former model, Playboy playmate, reality television star, and favorite tabloid subject Anna Nicole Smith was found unconscious in her hotel room at the Seminole Hard Rock Hotel and Casino in Hollywood, Florida. She was taken to Memorial Legal Hospital, where she was declared dead at age 39. Analysis of Smith’s blood postmortem revealed an array of prescribed medications. Most pronounced was a toxic level of the sedative chloral hydrate. A part of the contents of the toxicology report from Smith’s autopsy are shown here.

Although many of the drugs present were detected at levels consistent with typical doses of the prescribed medica- tions, it was their presence in combination with chloral hy- drate that exacerbated the toxic level of chloral hydrate. The lethal combination of these prescription drugs caused failure of both her circulatory and respiratory systems and resulted in her death. The investigators determined that the overdose of chloral hydrate and other drugs was accidental and not a suicide. This was due to the nonexcessive levels of most of the prescription medications and the discovery of a significant amount of chloral hydrate still remaining in its original con- tainer; had she intended to kill herself, she would have likely downed it all. Anna Nicole Smith was a victim of accidental overmedication.

Final Pathological Diagnoses

I. Acute Combined Drug Intoxication A. Toxic/legal drug: Chloral Hydrate (Noctec)

1. Trichloroethanol (TCE) 75 mg/L (active metabolite) 2. Trichloroacetic acid (TCA) 85 mg/L (inactive

metabolite) B. Therapeutic drugs:

1. Diphenhydramine (Benadryl) 0.11 mg/L 2. Clonazepam (Klonopin) 0.04 mg/L 3. Diazepam (Valium) 0.21 mg/L 4. Nordiazepam (metabolite) 0.38 mg/L 5. Temazepam (metabolite) 0.09 mg/L 6. Oxazepam 0.09 mg/L 7. Lorazepam 0.022 mg/L

C. Other noncontributory drugs present (atropine, topira- mate, ciprofloxacin, acetaminophen)

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acid A compound capable of donating a hydrogen ion (H1) to another compound.

base A compound capable of accepting a hydrogen ion (H1).

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ease. Conversely, a base is a compound that can pick up a hydrogen ion shed by an acid. The idea of acidity and basicity can be expressed in terms of a simple numerical value that relates to the concentration of the hydrogen ion (H1) in a liquid medium such as water. Chemists use the pH scale to do this. This scale runs from 0 to 14:

pH 5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

← Increasing acidity—Neutral—Increasing basicity →

Normally, water is neither acidic nor basic—in other words, it is neutral, with a pH of 7. However, when an acidic substance—for example, sulfuric acid or hydrochloric acid—is added to the water, it adds excess hydrogen ions, and the pH value becomes less than 7. The lower the number, the more acidic the water. Similarly, when a basic substance—for example, sodium hydroxide or ammonium hydroxide—is added to water, it removes hydrogen ions, thus making water basic. The more basic the water, the higher its pH value.

By controlling the pH of a water solution into which blood, urine, or tissues are dissolved, the toxicologist can conveniently control the type of drug that is recovered. For example, acidic drugs are easily extracted from an acidified water solution (pH less than 7) with organic solvents such as chloroform. Similarly, basic drugs are readily removed from a basic water solution (pH greater than 7) with organic solvents. This simple approach gives the toxicologist a general technique for extracting and categorizing drugs. Some of the more commonly encountered drugs may be classified as follows:

Acid Drugs Basic Drugs

Barbiturates Phencyclidine Acetylsalicylic acid (aspirin) Methadone Amphetamines Cocaine

SCREENING AND CONFIRMATION Once the specimen has been extracted and divided into acidic and basic fractions, the toxicologist can identify the drugs present. The strategy for identifying abused drugs entails a two-step approach: screening and confirmation (see Figure 12–7). A screening test is normally employed to give the analyst quick insight into the likelihood that a specimen contains a drug substance. This test allows a toxicologist to examine a large number of specimens within a short period of time for a wide range of drugs.

Acidic Drugs

Sample

SCREENING TEST Immunoassay

Gas chromatography Thin-layer chromatography

CONFIRMATION TEST Gas chromatography/mass spectrometry

Basic Drugs

Extraction at appropriate pH

FIGURE 12–7 Biological fluids and tissues are extracted for acidic and basic drugs by controlling the pH of a water solution in which they are dissolved. Once this is accomplished, the toxicologist analyzes for drugs by using screening and confirmation test procedures.

pH scale A scale used to express the basicity or acidity of a substance; a pH of 7 is neutral, whereas lower values are acidic and higher values are basic.

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Any positive results from a screening test are tentative at best and must be verified with a confirmation test.

The three most widely used screening tests are thin-layer chromatography (TLC), gas chromatography (GC), and immunoassay. The techniques of GC and TLC have already been described on pages 279–282 and 284–285, respectively. An immunoassay has proven to be a useful screening tool in toxicology laboratories. Its principles are very different from any of the analytical techniques we have discussed so far. Basically, immunoassay is based on specific drug antibody reactions. We will learn about this concept in Chapter 14. The primary advantage of im- munoassay is its ability to detect small concentrations of drugs in body fluids and organs. In fact, this technique provides the best approach for detecting the low drug levels normally associated with smoking marijuana.

The necessity of eliminating the possibility that a positive screening test may be due to a substance’s having a close chemical structure to an abused drug requires the toxicologist to follow up a positive screening test with a confirmation test. Because of the potential impact of the results of a drug finding on an individual, only the most conclusive confirmation proce- dures should be used. Gas chromatography/mass spectrometry is generally accepted as the confirmation test of choice. The combination of gas chromatography and mass spectrometry provides the toxicologist with a one-step confirmation test of unequaled sensitivity and specific- ity (see pages 291–292). As shown in Figure 12–8, the sample is separated into its components by the gas chromatograph. When the separated sample component leaves the column of the gas chromatograph, it enters the mass spectrometer, where it is bombarded with high-energy electrons. This bombardment causes the sample to break up into fragments, producing a frag- mentation pattern or mass spectrum for each sample. For most compounds, the mass spectrum represents a unique “fingerprint” pattern that can be used for identification.

There is tremendous interest in drug-testing programs conducted not only in criminal mat- ters but for industry and government as well. Urine testing for drugs is becoming common for job applicants and employees in the workplace. Likewise, the U.S. military has an extensive drug urine-testing program for its members. Many urine-testing programs rely on private laboratories to perform the analyses. In any case, when the test results form the basis for taking action against an individual, both a screening and confirmation test must be incorporated into the testing proto- col to ensure the integrity of the laboratory’s conclusions.

DETECTING DRUGS IN HAIR When a forensic toxicological examination on a living person is required, practicality limits available specimens to blood and urine. Most drugs remain in the bloodstream for about 24 hours; in urine, they normally are present up to 72 hours. However, it may be necessary to go further back in time to ascertain whether a subject has been abusing a drug. If so, the only viable alternative to blood and urine is head hair.

Hair is nourished by blood flowing through capillaries located close to the hair root. Drugs present in blood diffuse through the capillary walls into the base of the hair and become per- manently entrapped in the hair’s hardening protein structure. As the hair continues to grow, the

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FIGURE 12–8 The combination of the gas chromatograph and the mass spectrometer enables forensic toxicologists to separate the components of a drug mixture and provides specific identification of a drug substance.

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drug’s location on the hair shaft becomes a historical marker for delineating drug intake. Given that the average human head hair grows at the rate of 1 centimeter per month, analyzing seg- ments of hair for drug content may define the timeline for drug use, dating it back over a period of weeks, months, or even years, depending on the hair’s length.

However, caution is required in interpreting the timeline. The chronology of drug intake may be distorted by drugs penetrating the hair’s surface as a result of environmental exposure, or drugs may enter the hair’s surface through sweat. Nevertheless, drug hair analysis is the only viable approach for measuring long-term abuse of a drug.

Detecting Nondrug Poisons Although forensic toxicologists devote most of their efforts to detecting drugs, they also test for a wide variety of other toxic substances. Some of these are rare elements, not widely or commer- cially available. Others are so common that virtually everyone is exposed to nontoxic amounts of them every day.

HEAVY METALS The forensic toxicologist only occasionally encounters a group of poisons known as heavy metals. These include arsenic, bismuth, antimony, mercury, and thallium. To screen for many of these metals, the investigator may dissolve the suspect body fluid or tissue in a hydrochloric acid solution and insert a copper strip into the solution (the Reinsch test). The appearance of a silvery or dark coating on the copper indicates the presence of a heavy metal. Such a finding must be confirmed by the use of analytical techniques suitable for inorganic analysis—namely, atomic absorption spectrophotometry, emission spectroscopy, or X-ray diffraction.

CARBON MONOXIDE Unlike heavy metals, carbon monoxide still represents one of the most common poisons encountered in a forensic laboratory. When carbon monoxide enters the human body, it is primarily absorbed by the red blood cells, where it combines with hemoglobin to form carboxyhemoglobin. An average red blood cell contains about 280 million molecules of hemoglobin. Oxygen normally combines with hemoglobin, which transports the oxygen throughout the body. However, if a high percentage of the hemoglobin combines with carbon monoxide, not enough is left to carry sufficient oxygen to the tissues, and death by asphyxiation quickly follows.

There are two basic methods for measuring the concentration of carbon monoxide in the blood. Spectrophotometric methods examine the visible spectrum of blood to determine the amount of carboxyhemoglobin relative to oxyhemoglobin or total hemoglobin; also, a volume of blood can be treated with a reagent to liberate the carbon monoxide, which is then measured by gas chromatography.

The amount of carbon monoxide in blood is generally expressed as percent saturation. This represents the extent to which the available hemoglobin has been converted to carboxyhemoglobin. The transition from normal or occupational levels of carbon monoxide to toxic levels is not sharply defined. It depends, among other things, on the age, health, and general fitness of each individual. In a healthy middle-aged individual, a carbon monoxide blood saturation greater than 50–60 percent is considered fatal. However, in combination with alcohol or other depres- sants, fatal levels may be significantly lower. For instance, a carbon monoxide saturation of 35–40 percent may prove fatal in the presence of a blood-alcohol concentration of 0.20 percent w/v. Interestingly, chain smokers may have a constant carbon monoxide level of 8–10 percent from the carbon monoxide in cigarette smoke.

Inhaling automobile fumes is a relatively common way to commit suicide. A garden or vacuum cleaner hose is often used to connect the tailpipe with the vehicle’s interior, or the en- gine is allowed to run in a closed garage. A level of carbon monoxide sufficient to cause death accumulates in five to ten minutes in a closed single-car garage. IS

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The level of carbon monoxide in the blood of a victim found dead at the scene of a fire is significant in ascertaining whether foul play has occurred. High levels of carbon monoxide in the blood prove that the victim breathed the combustion products of the fire and was alive when the fire began. Many attempts at covering up a murder by setting fire to a victim’s house or car have been uncovered in this manner.

Significance of Toxicological Findings Once a drug is found and identified, the toxicologist assesses its influence on the behavior of the individual. Interpreting the results of a toxicology find is one of the toxicologist’s more difficult chores. Recall that many of the world’s countries have designated a specific blood- alcohol level at which an individual is deemed under the influence of alcohol. These levels were established as a result of numerous studies conducted over several years to measure the effects of alcohol levels on driving performance. However, no such legal guidelines are avail- able to the toxicologist who must judge how a drug other than alcohol affects an individual’s performance or physical state.

Joann Curley: Caught by a Hair A vibrant young woman named Joann Curley rushed to the Wilkes-Barre (Pennsylvania) General Hospital—her husband, Bobby, was having an attack and required immediate medi- cal attention. Bobby was experiencing a burning sensation in his feet, numbness in his hands, a flushed face, and intense sweating. He was diagnosed with Guillain-Barré syndrome, an acute inflammation of the nervous system that accounted for all of Bobby’s symptoms. After being discharged, Bobby ex- perienced another bout of debilitating pain and numbness. He was admitted to another hospital, the larger and more capable Hershey Medical Center in Hershey, Pennsylvania. There doc- tors observed extreme alopecia, or hair loss.

Test results of Bobby’s urine showed high levels of the heavy metal thallium in his body. Thallium, a rare and highly toxic metal that was used decades ago in substances such as rat poison and to treat ringworm and gout, was found in suffi- cient quantities to cause Bobby’s sickness. The use of thallium was banned in the United States in 1984. Now, at least, Bobby could be treated. However, before Bobby’s doctors could treat him for thallium poisoning, he experienced cardiac arrest and slipped into a coma. Joann Curley made the difficult decision to remove her husband of 13 months from life support equip- ment. He died shortly thereafter.

Bobby Curley was an electrician and, for five months be- fore his death, he worked in the chemistry department at nearby Wilkes University. Authorities suspected that Bobby had been accidentally exposed to thallium there among old chemicals and laboratory equipment. The laboratory was searched and several old bottles of powdered thallium salts were discov- ered in a storage closet. After testing of the air and surfaces, these were eliminated as possible sources for exposure. This

finding was supported by the discovery that none of Bobby’s co- workers had any thallium in their systems. The next most logical route of exposure was in the home; thus, the Curley kitchen was sampled. Of the hundreds of items tested, three thermoses were found to contain traces of thallium.

Investigators also learned that Bobby had changed his life insurance to list his wife, Joann, as the beneficiary of his $300,000 policy. Based on this information, police consulted a forensic toxicologist in an effort to glean as much from the physical evidence in Bobby Curley’s body as possible. The toxicologist conducted segmental analysis of Bobby’s hair, an analytical method based on the predictable rate of hair growth on the human scalp: an average of 1 centimeter per month. Bobby had approximately 5 inches (12.5 centimeters) of hair, which represents almost twelve months of hair growth. Each section tested represented a specific period of time in Bobby’s final year of his life.

The hair analysis proved that Bobby Curley was poisoned with thallium long before he began working at Wilkes Univer- sity. The first few doses were small, which probably barely made him sick at the time. Gradually, over a year or more, Bobby was receiving more doses of thallium until he finally succumbed to a massive dose three or four days before his death. After care- ful scrutiny of the timeline, investigators concluded that only Joann Curley had access to Bobby during each of these inter- vals. She also had motive, in the amount of $300,000.

Presented with the timeline and the solid toxicological evidence against her, Joann Curley pleaded guilty to murder. As part of her plea agreement, she provided a 40-page written confession of how she haphazardly dosed Bobby with some rat poison she found in her basement. She admitted that she murdered him for the money she would receive from Bobby’s life insurance policy.

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For many drugs, blood concentration levels are readily determined and can be used to es- timate the pharmacological effects of the drug on the individual. Often, when dealing with a living person, the toxicologist has the added benefit of knowing what a police officer may have observed about an individual’s behavior and motor skills, as well as the outcome of a drug influ- ence evaluation conducted by a police officer trained to be a drug recognition expert (discussed shortly). For a deceased person, drug levels in various body organs and tissues provide additional information about the individual’s state at the time of death. However, before conclusions can be drawn about a drug-induced death, other factors must also be considered, including the age, physical condition, and tolerance of the drug user.

With prolonged use of a drug, an individual may become less responsive to a drug’s effects and tolerate blood-drug concentrations that would kill a casual drug user. Therefore, knowledge of an individual’s history of drug use is important in evaluating drug concentrations. Another consideration is additive or synergistic effects of the interaction of two or more drugs, which may produce a highly intoxicated or comatose state even though none of the drugs alone is present at high or toxic levels. The combination of alcohol with tranquilizers or narcotics is a common example of a potentially lethal drug combination.

The presence of a drug present in urine is a poor indicator of how extensively an individual’s behavior or state is influenced by the drug. Urine is formed outside the body’s circulatory system, and consequently drug levels can build up in it over a long period. Some drugs are found in the urine one to three days after they have been taken and long after their effects on the user have disappeared. Nevertheless, the value of this information should not be discounted. Urine drug levels, like blood levels, are best used by law enforcement authorities and the courts to corrobo- rate other investigative and medical findings regarding an individual’s condition. Hence, for an individual who is arrested for suspicion of being under the influence of a drug, a toxicologist’s determinations supplement the observations of the arresting officer, including the results of a drug influence evaluation (discussed next).

For a deceased person, the responsibility for establishing a cause of death rests with the medical examiner or coroner. However, before a conclusive determination is made, the examin- ing physician depends on the forensic toxicologist to demonstrate the presence or absence of a drug or poison in the tissues or body fluids of the deceased. Only through the combined efforts of the toxicologist and the medical examiner (or coroner) can society be assured that death investi- gations achieve high professional and legal standards.

The Drug Recognition Expert Whereas recognizing alcohol-impaired performance is an expertise generally accorded to police officers by the courts, recognizing drug-induced intoxication is much more difficult and gener- ally not part of police training. During the 1970s, the Los Angeles Police Department developed and tested a series of clinical and psychophysical examinations that a trained police officer could use to identify and differentiate between types of drug impairment. This program has evolved into a national program to train police as drug recognition experts. Normally, a three- to five- month training program is required to certify an officer as a drug recognition expert (DRE).

The DRE program incorporates standardized methods for examining suspects to determine whether they have taken one or more drugs. The process is systematic and standard; to ensure that each subject has been tested in a routine fashion, each DRE must complete a standard Drug Influence Evaluation form (see Figure 12–9). The entire drug evaluation takes approximately 30 to 40 minutes. The components of the 12-step process are summarized in Table 12–1.

The DRE evaluation process can suggest the presence of the following seven broad catego- ries of drugs:

1. Central nervous system depressants 2. Central nervous system stimulants 3. Hallucinogens 4. Dissociative anesthetics (includes phencyclidine and its analogs) 5. Inhalants 6. Narcotic analgesics 7. Cannabis

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FIGURE 12–9 Drug Influence Evaluation form.

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The DRE program is not designed to be a substitute for toxicological testing. The toxicolo- gist can often determine that a suspect has a particular drug in his or her body. But the toxicolo- gist often cannot infer with reasonable certainty that the suspect was impaired at a specific time. On the other hand, the DRE can supply credible evidence that the suspect was impaired at a spe- cific time and that the nature of the impairment was consistent with a particular family of drugs. But the DRE program usually cannot determine which specific drug was ingested. Proving drug intoxication requires a coordinated effort and the production of competent data from both the DRE and the forensic toxicologist.

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TABLE 12–1 Components of the Drug Recognition Process

1. The Breath-Alcohol Test. By obtaining an accurate and immediate measurement of the suspect’s blood-alcohol concentration, the drug recognition expert (DRE) can determine whether alcohol may be contributing to the suspect’s observable impairment and whether the concentration of alcohol is sufficient to be the sole cause of that impairment.

2. Interview with the Arresting Officer. Spending a few minutes with the arresting officer often enables the DRE to determine the most promising areas of investigation.

3. The Preliminary Examination. This structured series of questions, specific observations, and simple tests provides the first opportunity to examine the suspect closely. It is designed to determine whether the suspect is suffering from an injury or from another condition unrelated to drug consumption. It also affords an opportunity to begin assessing the suspect’s appearance and behavior for signs of possible drug influence.

4. The Eye Examination. Certain categories of drugs induce nystagmus, an involuntary, spasmodic motion of the eyeball. Nystagmus is an indicator of drug-induced impairment. The inability of the eyes to converge toward the bridge of the nose also indicates the possible presence of certain types of drugs.

5. Divided-Attention Psychophysical Tests. These tests check balance and physical orientation and include the walk and turn, the one-leg stand, the Romberg balance, and the finger-to-nose.

6. Vital Signs Examinations. Precise measurements of blood pressure, pulse rate, and body temperature are taken. Certain drugs elevate these signs; others depress them.

7. Dark Room Examinations. The size of the suspect’s pupils in room light, near-total darkness, indirect light, and direct light is checked. Some drugs cause the pupils to either dilate or constrict.

8. Examination for Muscle Rigidity. Certain categories of drugs cause the muscles to become hypertense and quite rigid. Others may cause the muscles to relax and become flaccid.

9. Examination for Injection Sites. Users of certain categories of drugs routinely or occasionally inject their drugs. Evidence of needle use may be found on veins along the neck, arms, and hands.

10. Suspect’s Statements and Other Observations. The next step is to attempt to interview the suspect concerning the drug or drugs he or she has ingested. Of course, the interview must be conducted in full compliance with the suspect’s constitutional rights.

11. Opinions of the Evaluator. Using the information obtained in the previous ten steps, the DRE can make an informed decision about whether the suspect is impaired by drugs and, if so, what category or combination of categories is the probable cause of the impairment.

12. The Toxicological Examination. The DRE should obtain a blood or urine sample from the suspect for laboratory analysis in order to secure scientific, admissible evidence to substantiate his or her conclusions.

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Toxicologists detect and identify the presence of drugs and poisons in body fluids, tissues, and organs. A major branch of forensic toxicology deals with the measurement of alcohol in the body for matters that pertain to violations of criminal law. Alcohol appears in the blood within minutes after it has been taken by mouth and slowly increases in concentration while it is being absorbed from the stomach and the small intestine into the bloodstream. When all the alcohol has been absorbed, a maximum alcohol level is reached in the blood and the postabsorption period begins. Then the alcohol concentration slowly decreases until a zero level is again reached. Alcohol is eliminated from the body through oxidation and excretion. Oxidation takes place almost entirely in the liver, whereas al- cohol is excreted unchanged in the breath, urine, and perspira- tion. The extent to which an individual is under the influence of alcohol is usually determined by measuring the quantity of alcohol in the blood or the breath. Breath testers that oper- ate on the principle of infrared light absorption are becoming increasingly popular within the law enforcement community.

Many types of breath testers analyze a set volume of breath. The sampled breath is exposed to infrared light. The degree of interaction of the light with alcohol in the breath sample allows the instrument to measure a blood-alcohol con- centration in breath. These breath-testing devices operate on the principle that the ratio between the concentration of alco- hol in deep-lung or alveolar breath and its concentration in blood is fixed. Most breath-test devices have set the ratio of alcohol in the blood to alcohol in alveolar air at 2,100 to 1.

Law enforcement officers typically use field sobriety tests to estimate a motorist’s degree of physical impairment by alcohol and whether an evidential test for alcohol is justi- fied. The horizontal-gaze nystagmus test, walk and turn, and

the one-leg stand are all reliable and effective psychophysical tests.

Gas chromatography is the most widely used approach for determining alcohol levels in blood. Blood must always be drawn under medically accepted conditions by a qualified individual. A nonalcoholic disinfectant must be applied before the suspect’s skin is penetrated with a sterile needle or lancet. Once blood is removed from an individual, it is best preserved sealed in an airtight container after adding an anticoagulant and a preservative.

The forensic toxicologist must devise an analytical scheme to detect, isolate, and identify toxic drug substances. Once the drug has been extracted from appropriate biological fluids, tissues, and organs, the forensic toxicologist can iden- tify the drug substance. The strategy for identifying abused drugs entails a two-step approach: screening and confirma- tion. A screening test gives the analyst quick insight into the likelihood that a specimen contains a drug substance. Positive results from a screening test are tentative at best and must be verified with a confirmation test. The most widely used screen- ing tests are thin-layer chromatography, gas chromatography, and immunoassay. Gas chromatography/mass spectrometry is generally accepted as the confirmation test of choice. Once the drug is extracted and identified, the toxicologist may be required to judge the drug’s effect on an individual’s natural performance or physical state. The Drug Recognition Expert program incorporates standardized methods for examining automobile drivers suspected of being under the influence of drugs. But the DRE program usually cannot determine which specific drug was ingested. Hence, reliable data from both the DRE and the forensic toxicologist are required to prove drug intoxication.

chapter summary

review questions

1. The most heavily abused drug in the Western world is ___________.

2. True or False: Toxicologists are employed only by crime laboratories. ___________

3. The amount of alcohol in the blood (is, is not) directly proportional to the concentration of alcohol in the brain.

4. True or False: Blood levels have become the accepted standard for relating alcohol intake to its effect on the body. ___________

5. Alcohol consumed on an empty stomach is absorbed (faster, slower) than an equivalent amount of alcohol taken when there is food in the stomach.

6. Under normal drinking conditions, alcohol concentra- tion in the blood peaks in ___________ to ___________ minutes.

7. In the postabsorption period, alcohol is distributed uni- formly among the ___________ portions of the body.

8. Alcohol is eliminated from the body by ___________ and ___________.

9. Ninety-five to 98 percent of the alcohol consumed is ___________ to carbon dioxide and water.

10. Oxidation of alcohol takes place almost entirely in the ___________.

11. The amount of alcohol exhaled in the ___________ is directly proportional to the concentration of alcohol in the blood.

12. Alcohol is eliminated from the blood at an average rate of ___________ percent w/v.

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living person suspected of being under the influence of a drug.

26. A large number of drugs can be classified chemically as ___________ and ___________.

27. Water with a pH value (less, greater) than 7 is basic.

28. Barbiturates are classified as ___________ drugs.

29. Drugs are extracted from body fluids and tissues by carefully controlling the ___________ of the medium in which the sample has been dissolved.

30. The technique of ___________ is based on specific drug antibody reactions.

31. Both ___________ and ___________ tests must be in- corporated into the drug-testing protocol of a toxicology laboratory to ensure the correctness of the laboratory’s conclusions.

32. The gas ___________ combines with hemoglobin in the blood to form carboxyhemoglobin, thus interfering with the transportation of oxygen in the blood.

33. The amount of carbon monoxide in blood is usually ex- pressed as ___________.

34. True or False: Blood levels of drugs can alone be used to draw definitive conclusions about the effects of a drug on an individual. ___________

35. Interaction of alcohol and barbiturates in the body can produce a(n) ___________ effect.

36. The level of a drug present in the urine is by itself a (good, poor) indicator of how extensively an individual is affected by a drug.

37. Urine and blood drug levels are best used by law en- forcement authorities and the courts to ___________ other investigative and medical findings pertaining to an individual’s condition.

38. The ___________ program incorporates standardized methods for examining suspects to determine whether they have taken one or more drugs.

13. Alcohol is absorbed into the blood from the ___________ and ___________.

14. Most modern breath testers use ___________ radiation to detect and measure alcohol in the breath.

15. To avoid the possibility of “mouth alcohol,” the opera- tor of a breath tester must not allow the subject to take any foreign materials into the mouth for ___________ minutes before the test.

16. Alcohol can be separated from other volatiles in blood and quantitated by the technique of ___________.

17. Roadside breath testers that use a(n) ___________ de- tector are becoming increasingly popular with the law enforcement community.

18. True or False: Portable handheld roadside breath testers for alcohol provide evidential test results. ___________

19. Usually, when a person’s blood-alcohol concentration is in the range of 0.10 percent, horizontal-gaze nystagmus begins before the eyeball has moved ___________ de- grees to the side.

20. When drawing blood for alcohol testing, the sus- pect’s skin must first be wiped with a(n) ___________ disinfectant.

21. Failure to add a preservative, such as sodium fluoride, to blood removed from a living person may lead to a(n) (decline, increase) in alcohol concentration.

22. Most states have established ___________ percent w/v as the impairment limit for blood-alcohol concentration.

23. In the case of ___________, the Supreme Court ruled that taking nontestimonial evidence, such as a blood sample, did not violate a suspect’s Fifth Amendment rights.

24. Heroin is changed upon entering the body into ___________.

25. The body fluids ___________ and ___________ are both desirable for the toxicological examination of a

review questions for inside the science

1. A(n) ___________ carries blood away from the heart; a(n) ___________ carries blood back to the heart.

2. The ___________ artery carries deoxygenated blood from the heart to the lungs.

3. Alcohol passes from the blood capillaries into the ___________ sacs in the lungs.

4. One milliliter of blood contains the same amount of al- cohol as approximately ___________ milliliters of al- veolar breath.

5. When alcohol is being absorbed into the blood, the alco- hol concentration in venous blood is (higher, lower) than that in arterial blood.

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application and critical thinking

1. Answer the following questions about driving risk as- sociated with drinking and blood-alcohol concentration:

a. Randy is just barely legally intoxicated. How much more likely is he to have an accident than someone who is sober?

b. Marissa, who has been drinking, is 15 times as likely to have an accident as her sober friend, Chris- tine. What is Marissa’s approximate blood-alcohol concentration?

c. After several drinks, Charles is ten times as likely to have an accident as a sober person. Is he more or less intoxicated than James, whose blood-alcohol level is 0.10?

d. Under the original blood-alcohol standards rec- ommended by NHTSA, a person considered just barely legally intoxicated was how much more likely to have an accident than a sober individual?

2. Following is a description of four individuals who have been drinking. Rank them from highest to lowest blood- alcohol concentration:

a. John, who weighs 200 pounds and has consumed eight 8-ounce drinks on a full stomach

b. Frank, who weighs 170 pounds and has consumed four 8-ounce drinks on an empty stomach

c. Gary, who weighs 240 pounds and has consumed six 8-ounce drinks on an empty stomach

d. Stephen, who weighs 180 pounds and has consumed six 8-ounce drinks on a full stomach

3. Following is a description of four individuals who have been drinking. In which (if any) of the following coun- tries would each be considered legally drunk: the United States, Australia, Sweden?

a. Bill, who weighs 150 pounds and has consumed three 8-ounce drinks on an empty stomach

b. Sally, who weighs 110 pounds and has consumed three 8-ounce drinks on a full stomach

c. Rich, who weighs 200 pounds and has consumed six 8-ounce drinks on an empty stomach

d. Carrie, who weighs 140 pounds and has consumed four 8-ounce drinks on a full stomach

4. You are a forensic scientist who has been asked to test two blood samples. You know that one sample is sus- pected of containing barbiturates and the other contains no drugs; however, you cannot tell the two samples apart. Describe how you would use the concept of pH to determine which sample contains barbiturates. Explain your reasoning.

5. You are investigating an arson scene and you find a corpse in the rubble, but you suspect that the victim did not die as a result of the fire. Instead, you suspect that the victim was murdered earlier, and that the blaze was started to cover up the murder. How would you go about determining whether the victim died before the fire?

further references

Benjamin, David M., “Forensic Pharmacology,” in R. Saferstein, ed., Forensic Science Handbook, vol. 3, 2nd ed. Upper Saddle River, N.J.: Prentice Hall, 2010.

Caplan, Y. H., and J. R. Zettl, “The Determination of Alcohol in Blood and Breath,” in R. Saferstein, ed., Forensic Science Handbook, vol. 1, 2nd ed. Upper Saddle River, N.J.: Prentice Hall, 2002.

Couper, F. J., and B. K. Logan, Drugs and Human Performance. Washington, D.C.: National Highway Traffic Safety Administration, 2004, http://www.nhtsa .dot.gov/people/injury/research/job185drugs/ technical-page.htm

Garriott, James C., ed., Medicolegal Aspects of Alcohol, 5th ed. Tucson, Ariz.: Lawyers & Judges, 2009.

Levine, B., ed., Principles of Forensic Toxicology, 3rd ed. Washington, D.C.: AACC Press, 2006.

Ropero-Miller, J. D., and B. A. Goldberger, eds., Handbook of Workplace Drug Testing, 2nd ed. Washington, D.C.: AACC Press, 2009.

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The Green River Killer

headline news

This case takes its name from the Green River, which flows through Washington state and empties into Puget Sound in Seattle. In 1982, within six months the bodies of five females were discovered in or near the river. Most of the victims were known prostitutes who were strangled and apparently raped. As police focused their attention on an area known as Sea-Tac Strip, a haven for prostitutes, girls mysteriously disappeared with increasing frequency. By the end of 1986, the body count in the Seattle region rose to 40, all of whom were believed to have been murdered by the Green River Killer. As the investigation pressed on into 1987, the police renewed their interest in one suspect, Gary Ridgway, a local truck painter. Interestingly, in 1984 Ridgway had passed a lie detector test. Now with a search warrant in hand, police searched the Ridgway residence and also obtained hair and saliva samples from Ridgway. Again, insufficient evidence caused Ridgway to be released from custody. However, as the investigation proceeded, a DNA link between Ridgway and his victims eluded investigators. Ultimately, a careful microscopic search of

Ridgway’s clothing revealed the presence of paint spheres of various colors, which compared to spheres on the clothing of six

of the victims. The paint was microscopically and chemically identified as Imron, a high-end specialty paint that was manufactured before 1984. This product had been used at

the truck plant where Ridgway worked and was identified as dried paint spheres emanating from a spray paint. Two of the victims were further linked to Ridgway through DNA, further solidifying the case against Ridgway. Ridgway avoided the death penalty by confessing to the murders of 48 women.

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After studying this chapter you should be able to: Describe the usefulness of trace elements for forensic comparison of various types of physical evidence

Distinguish continuous and line emission spectra

Understand the parts of a simple emission spectrograph

Define and distinguish protons, neutrons, and electrons

Define and distinguish atomic number and atomic mass number

Appreciate the phenomenon of how an atom absorbs and releases energy in the form of light

Explain the concept of an isotope

Understand how elements can be made radioactive

List the most useful examinations for performing a forensic comparison of paint

Describe the proper collection and preservation of forensic paint evidence

List the important forensic properties of soil

Describe the proper collection of soil evidence

metals, paint, and soil

alpha particle atomic mass atomic number beta particle continuous spectrum electron electron orbital emission spectrum excited state gamma ray isotope line spectrum mineral neutron nucleus proton pyrolysis radioactivity

KEY TERMS

chapter 13

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Forensic Analysis of Trace Elements Considering that most of our raw materials originate from the earth’s crust, it is not surprising that they are rarely obtained in pure form; instead, they include numerous elemental impurities that usually have to be eliminated through industrial processing. However, in most cases it is not economically feasible to completely exclude all such minor impurities, especially when their presence will have no effect on the appearance or performance of the final product. For this rea- son, many manufactured products, and even most natural materials, contain small quantities of elements present in concentrations of less than 1 percent.

For the criminalist, the presence of trace elements is particularly useful because they pro- vide “invisible” markers that may establish the source of a material or at least provide additional points for comparison. Glass fragments represent a valuable type of trace evidence; however, generally because of their minute size they present the criminalist with two distinct issues. First is classifying the type of glass being examined. Three types of glass are normally encountered as forensic evidence; float glass (windows, windshields), container glass (bottles, jars), and bo- rosilicates (kitchenware). Figure 13–1 depicts an elemental analysis comparing three different glasses and clearly shows how they are distinguished by the intensity of the peaks associated with Boron (B) and Magnesium (Mg).

Similarly, the comparison of trace elements present in glass may provide particularly mean- ingful data with respect to source or origin. Technological advances in the manufacture of glass has led to more uniformity in the final product. Unfortunately, this has the consequence of dimin- ishing the value of the most important comparative physical property—refractive index. Fortu- nately, minor variations in the chemical composition of glass remain between and within batches because of the presence of natural contaminants in raw materials. Up to 25 different elements

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FIGURE 13–1 The presence of trace elements in glass as shown above can be used to identify glass types.

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METALS, PAINT, AND SOIL 329

have been identified in glass. The forensic discrimination associated with glass comparisons can now be enhanced by combining elemental analysis with refractive index (see Figure 13–2). Forensic investigators have also examined the evidential value of trace elements present in soil, fibers, and paint, as well as in all types of metallic objects. One example of this application oc- curred with the examination of the bullet and bullet fragments recovered after the assassination of President Kennedy.

Evidence in the Assassination of President Kennedy Ever since President Kennedy was killed in 1963, questions have lingered about whether Lee Harvey Oswald was part of a conspiracy to assassinate the president or, as the Warren Commis- sion concluded, a lone assassin. In arriving at its conclusions, the Warren Commission recon- structed the crime as follows: Oswald fired three shots from behind the president while positioned in the Texas School Book Depository building. The president was struck by two bullets, with one bullet totally missing the president’s limousine. One bullet hit the president in the back, exited his throat, and then went on to strike Governor Connally, who was sitting in a jump seat in front of the president. The bullet hit Connally first in his back, then exited his chest, struck his right wrist,

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FIGURE 13–2 The presence of trace elements in glass as shown here can be used to discriminate between glass particles that are indistinguishable by other test protocols, such as refractive index.

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330 CHAPTER 13

and temporarily lodged in his left thigh. This bullet was later found on the governor’s stretcher at the hospital. A second bullet in the skull fatally wounded the president (see Figure 13–3).

In a room at the Texas School Book Depository, a 6.5-mm Mannlicher-Carcano military rifle was found with Oswald’s palm print on it. Also found were three spent 6.5-mm Western Cartridge Co./Mannlicher-Carcano (WCC/MC) cartridge cases. Oswald, an employee of the de- pository, had been seen there that morning and also a few minutes after the assassination, disap- pearing soon thereafter. He was apprehended a few miles from the depository nearly two hours after the shooting.

Critics of the Warren Commission have long argued that evidence exists that would prove Oswald did not act alone. Eyewitness accounts and acoustical data interpreted by some experts have been used to advocate the contention that someone else fired at the president from a region in front of the limousine (the so-called grassy knoll). Furthermore, it is argued that the Warren Commission’s reconstruction of the crime relied on the assumption that only one bullet caused both the president’s throat wound and Connally’s back wound. Critics contend that such damage would have deformed and mutilated a bullet. Instead, the recovered bullet showed some flatten- ing, no deformity, and only about 1 percent weight loss.

In 1977, at the request of the U.S. House of Representatives Select Committee on Assassina- tions, the bullet taken from Connally’s stretcher along with bullet fragments recovered from the car and various wound areas were examined for trace element levels.

Lead alloys used for the manufacture of bullets contain an assortment of trace elements. For example, antimony is often added to lead as a hardening agent; copper, bismuth, and silver are other trace elements commonly found in bullet lead. In this case, the bullet and bullet fragments were compared for their antimony and silver content. Previous studies had amply demonstrated that the levels of these two elements are particularly important for characterizing WCC/MC bullets. Bullet lead from this type of ammunition ranges in antimony concentration from 20 to 1,200 parts per million (ppm) and 5 to 15 ppm in silver content.

FIGURE 13–3 President John F. Kennedy, Governor John Connally of Texas, and Mrs. Jacqueline Kennedy ride through Dallas moments before the assassination.

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As can be seen in Table 13–1, the samples designated Q1 and Q9 (the Connally stretcher bullet and fragments from Connally’s wrist, respectively) are indistinguishable from one another in antimony and silver content. The samples Q2, Q4, Q5, and Q14 (Q4 and Q5 being fragments from Kennedy’s brain, and Q2 and Q14 being fragments recovered from two different areas in the car) also are indistinguishable in antimony and silver content but are different from Q1 and Q9.

The conclusions derived from studying these results are as follows:

1. There is evidence of only two bullets—one composed of 815 ppm antimony and 9.3 ppm silver, the other composed of 622 ppm antimony and 8.1 ppm silver.

2. Both bullets have a composition highly consistent with WCC/MC bullet lead, although other sources cannot entirely be ruled out.

3. The bullet found on the Connally stretcher also damaged Connally’s wrist. The absence of bullet fragments from the back wounds of Kennedy and Connally prevented any effort at linking these wounds to the stretcher bullet.

None of these conclusions can totally verify the Warren Commission’s reconstruction of the assassination, but the results are at least consistent with the commission’s findings that two WCC/MC bullets struck the occupants of the President’s limousine. Further, in 2003, an ABC television broadcast showed the results of a ten-year 3-D computer animation study of the events of November 22, 1963. The animation graphically showed that the bullet wounds were com- pletely consistent with Kennedy’s and Connally’s positions at the time of shooting, and that by following the bullet’s trajectory backward they could be found to have originated from a narrow cone including only a few windows of the sixth floor of the Texas School Book Depository.

The Emission Spectrum of Elements We have already observed that molecules can readily be characterized by their selective absorp- tion of ultraviolet, visible, or infrared radiation. Equally significant to the analytical chemist is the knowledge that elements also selectively absorb and emit light. These observations form the basis of an important analytical technique designed to determine the elemental composition of materials—emission spectroscopy.

TYPES OF SPECTRA The statement that elements emit light should not come as a total surprise, for one need only observe the common tungsten incandescent lightbulb or the glow of a neon light to confirm this observation. When the light emitted from a bulb or from any other light source is passed through a prism, it is separated into its component colors or frequencies. The resulting display of colors is called an emission spectrum. When sunlight or the light from an incandescent bulb is passed through a prism, a range of rainbow colors is produced. This emission spectrum is called a continuous spectrum because all the colors merge or blend into one another to form a continuous band. However, not all light sources produce such a spectrum. For example, if the light from a sodium lamp, a mercury arc lamp, or a neon light were passed through a prism, the resultant spectrum would consist not of a continuous band but of several individual colored

TABLE 13–1 Bullet and Bullet Fragments Examined in the Kennedy Assassination Investigation

Sample Description

Q1 Connally stretcher bullet Q9 Fragments from Connally’s wrist Q2 Large fragment from car Q4, Q5 Fragments from Kennedy’s brain Q14 Small fragment found in car

Elemental analysis classified the bullets and fragments into two distinctly different groups. Q1 and Q9 were of similar composition containing 815 ppm1 antimony and 9.3 ppm of silver, respectively. Q2, Q4, Q5, And Q14 fell into a second group comprised of 622 ppm of antimony and 8.1 ppm of silver, respectively. All the samples examined were consistent with WCC/MC bullet lead, although other sources could not be entirely ruled out. 1 One part per million equals 0.0001 percent.

emission spectrum Light emitted from a source and separated into its component colors or frequencies.

continuous spectrum A type of emission spectrum show- ing a continuous band of colors all blending into one another.

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lines separated by dark spaces. Here, each line represents a definite wavelength or frequency of light that is separate and distinct from all others present in the spectrum. This type of spectrum is called a line spectrum. Figure 13–4 shows the line spectra of three elements.

If a solid or liquid is vaporized and “excited” by exposure to a high temperature, each ele- ment present emits light composed of select frequencies that are characteristic of the element. This spectrum is in essence a “fingerprint” of an element and offers a practical method of iden- tification. Sodium vapor, for example, always shows the same line spectrum, which differs from the spectrum of all other elements.

Atomic Structure Any proposed theory that attempts to explain the origin of emission spectra must relate to the fundamental structure of the element—the atom. Scientists now know that the atom is composed of even more elementary particles that are collectively known as subatomic particles. The most important subatomic particles are the proton, electron, and neutron. The masses of the proton and neutron are each about 1,837 times the mass of an electron. The proton has a positive electri- cal charge; the electron has a negative charge equal in magnitude to that of the proton; and the neutron is a neutral particle having neither a positive nor a negative charge. The properties of the proton, neutron, and electron are summarized in the following table:

line spectrum A type of emission spectrum show- ing a series of lines separated by black areas; each line represents a definite wavelength or frequency.

Hydrogen

Helium

Mercury

FIGURE 13–4 Some characteristic emission spectra.

proton A positively charged particle that is one of the basic structures in the nucleus of an atom.

electron A negatively charged particle that is one of the fundamental structural units of the atom.

neutron A particle with no electrical charge that is one of the basic structures in the nucleus of an atom.

Particle Symbol Relative Mass Electrical Charge

Proton P 1 11 Neutron n 1 0 Electron e 1/1,837 –1

1 Actually, the electrons are moving so rapidly around the nucleus as to best be visualized as being in the form of an electron cloud spread out over the surface of the atom.

nucleus The core of an atom, containing the protons and neutrons.

A popular descriptive model of the atom, and the one that will be adopted for the purpose of this discussion, pictures an atom as consisting of electrons orbiting around a central nucleus—an image that is analogous to our solar system, in which the planets revolve around the sun.1 The nucleus of the atom is composed of positively charged protons and neutrons, which have no charge. Because the atom has no net electrical charge, the number of protons must always be equal to the number of negatively charged electrons in orbit around the nucleus.

With this knowledge, we can now begin to describe the atomic structure of the elements; for example, hydrogen has a nucleus consisting of one proton and no neutrons, and it has one orbit- ing electron. Helium has a nucleus comprising two protons and two neutrons, with two electrons in orbit around the nucleus (see Figure 13–5).

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Hydrogen

2P 2n

Helium

FIGURE 13–5 The atomic structures of hydrogen and helium.

lens and focused onto a prism that disperses it into component frequencies. The separated frequen- cies are then directed toward a photographic plate, where they are recorded as line images. Normally, a specimen consists of numerous elements; hence, the typical emission spectrum contains many lines. Each element present in the spectrum can be identified when it is compared to a standard chart that shows the position of the principal spectral lines of all the elements. However, forensic analysis more commonly requires simply a rapid comparison of the elemen- tal composition of two or more specimens. This can readily be accomplished when the emission spectra are matched line for line, an approach illustrated in the figure, in which the emission spectra of two paint chips are shown to be comparable.

Carbon Arc Emission Spectrometry

An emission spectrograph is an instrument used to obtain and record the line spectra of elements. Es- sentially, this instrument requires a means for vapor- izing and exciting the atoms of elements so that they emit light, a means for separating this light into its component frequencies, and a means of recording the resultant spectrum. A simple carbon arc emission spectrograph is depicted in the figure.

The specimen under investigation is excited when it is inserted between two carbon electrodes through which a direct current arc is passed. The arc produces enough heat to vaporize and excite the specimen’s atoms. The resultant emitted light is collected by a

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Lens Prism Photographic

plate

Sample between carbon electrodes

Parts of a simple carbon arc emission spectrograph.

A comparison of paint chips 1 and 2 by emission spectrographic analysis. A line-for-line comparison shows that the paints have the same elemental composition.

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The behavior and properties that distinguish one element from another must be related to the differences in the atomic structure of each element. One such distinction is that each element pos- sesses a different number of protons. This number is called the atomic number of the element. As we look back at the periodic table illustrated in Figure 9–1, we see that the elements are numbered consecutively. Those numbers represent the atomic number or number of protons associated with each element. An element is therefore a collection of atoms that all have the same number of protons. Thus, each atom of hydrogen has one and only one proton, each atom of helium has 2 protons, each atom of silver has 47 protons, and each atom of lead has 82 protons in its nucleus.

Inductively Coupled Plasma Emission Spectrometry (ICP) Carbon arc emission spectrometry has been supplanted by inductively coupled plasma (ICP) emission spectrometry. Like the former, ICP identifies and measures elements through light en- ergy emitted by excited atoms. However, instead of using an electrical arc, the atoms are ex- cited by placing the sample in a hot plasma torch. The torch is designed as three concentric quartz tubes through which argon gas flows. A radio frequency (RF) coil that carries a current is wrapped around the tubes. The RF current creates an intense magnetic field.

THE ICP PROCESS The process begins when a high-voltage spark is applied to the argon gas flowing through the torch. This strips some electrons from the argon atoms. These electrons are then caught and accelerated in the magnetic field such that they collide with other argon atoms, stripping off still more electrons. The collision of electrons and argon atoms continues in a chain reaction, breaking down the gas into argon atoms, argon ions, and electrons and forming an inductively coupled plasma discharge. The discharge is sustained by RF energy that is continuously transferred to it from the coil. The plasma discharge acts like an intense continuous

and it quickly falls back to its original energy level. As the electron falls back, it releases energy. An emis- sion spectrum testifies to the fact that this energy loss comes about in the form of light emission, as shown in the figure. The frequency of light emitted is again determined by the relationship E 5 hf, where E is the energy difference between the upper and lower en- ergy levels, h is a constant known as Planck’s constant and f is the frequency of emitted light. Because each element has its own characteristic set of energy levels, each emits a unique set of frequency values. The emis- sion spectrum thus provides a “picture” of the energy levels that surround the nucleus of each element.

Thus, we see that as far as atoms are concerned, energy can be put into the atom at the same time energy is given off; what goes in must come out. The chemist can study the atom using either approach.

The Origin of Emission Spectra

To explain the origin of atomic spectra, our atten- tion must now focus on the electron orbitals of the atom. As electrons move around the nucleus, they are confined to a path from which they cannot stray. This orbital path is associated with a definite amount of energy and is therefore called an energy level. Each element has its own set of characteristic energy levels at varying distances from the nucleus. Some levels are occupied by electrons; others are empty.

An atom is in its most stable state when all of its electrons are positioned in their lowest possible en- ergy orbitals in the atom. When an atom absorbs en- ergy, such as heat or light, its electrons are pushed into higher-energy orbitals. In this condition, the atom is in an excited state. However, because energy lev- els have fixed values, only a definite amount of energy can be absorbed in moving an electron from one level to another. This is a most important observation, for it means that atoms absorb only a definite value of energy, and all other energy values will be excluded. In the same manner, if atoms are exposed to intense heat, enough energy is generated to push electrons into unoccupied higher-energy orbitals. Normally, the electron does not remain in this excited state for long,

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electron orbital The path of electrons as they move around the nuclei of atoms; each orbital is associated with a particu- lar electronic energy level.

excited state The state in which an atom absorbs energy and an electron moves from a lower to a higher energy level.

(a) (b)

(a) The absorption of light by an atom, causing an electron to jump into a higher orbital. (b) The emission of light by an atom, caused by an electron falling back to a lower orbital.

atomic number The number of protons in the nucleus of an atom; each element has its own unique atomic number.

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flame, generating extremely high temperatures in the range of 7,000–10,000°C. The sample, in the form of an aerosol, is then introduced into the hot plasma, where it collides with the energetic argon electrons, generating charged particles (ions) that emit light of characteristic wavelengths corresponding to the identity of the elements present (see Figure 13–6).

Isotopes and Radioactivity Until now, our discussion of subatomic particles has been limited to the proton and electron. However, to understand the principles of nuclear chemistry, we must look at the other important subatomic particle, the neutron. Although the atoms of a single element must have the same number of protons, nothing prevents them from having different numbers of neutrons. The total number of protons and neutrons in a nucleus is known as the atomic mass number.

Atoms with the same number of protons but differing solely in the number of neutrons are called isotopes. For example, hydrogen consists of three isotopes; besides ordinary hydrogen, which has one proton and no neutrons, two other isotopes exist, deuterium and tritium. Deuterium (or heavy hydrogen) also has one proton but contains one neutron as well. Tritium has one proton

Sample aerosol

Coil

Ions

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+ ++

++ +

Rf generator

Plasma discharge

FIGURE 13–6 The creation of charged particles in the torch of an ICP discharge.

class physical evidence—how can a forensic analyst explain to a jury that such a finding has meaningful consequences to a criminal inquiry without being able to provide statistical or probability data to sup- port such a contention? Furthermore, the creation of meaningful databases to statistically define the significance of bullets compared by their elemental profiles is currently an unrealistic undertaking. Nev- ertheless, the significant diversity of bullet lead com- positions in our population, like other class evidence such as fibers, hairs, paint, plastics, and glass, makes their chance occurrence at a crime scene and sub- sequent link to a defendant a highly unlikely event. However, care must be taken to avoid giving the trier of fact the impression that elemental profiles consti- tute a definitive match. Given the millions of bullets produced each year, one cannot conclusively rule out the possibility of a coincidental match with a non- case-related bullet.

ICP Analysis of Bullets

Mutilated bullets often are not suitable for traditional microscopic comparisons against an exemplar test- fired bullet. In such situations, ICP has been used to obtain an elemental profile of the questioned bullet fragment for comparison against an unfired bullet, generally found in the possession of the suspect. For a number of years forensic scientists have taken advan- tage of significant compositional differences among lead sources for the manufacture of lead-based bullets. Compositional differences in the trace elements that constitute lead bullets are typically reflected in the cop- per, arsenic, silver, antimony, bismuth, cadmium, and tin profiles of lead bullets. When two or more bullets have comparable elemental compositions, evidence of their similarity may be offered in a court of law.

In this respect, the comparison of lead bullets faces the same quandary as most common types of

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atomic mass The sum of the number of protons and neutrons in the nucleus of an atom.

isotope An atom differing from another atom of the same element in the number of neutrons in its nucleus.

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and two neutrons in its nucleus. The atomic structures of these isotopes are shown in Figure 13–7. Therefore, all the isotopes of hydrogen have an atomic number of 1 but differ in their atomic mass numbers. Hydrogen has an atomic mass number of 1, deuterium a mass of 2, and tritium a mass of 3. Ordinary hydrogen makes up 99.98 percent of all the hydrogen atoms found in nature.

Like hydrogen, most elements are known to have two or more isotopes. Tin, for example, has ten isotopes. Many of these isotopes are quite stable, and for all intents and purposes, the isotopes of any one element have indistinguishable properties. Others, however, are not as stable and decompose with time by a process known as radioactive decay. Radioactivity is the emis- sion of radiation that accompanies the spontaneous disintegration of unstable nuclei. Radioactivity is actually composed of three types of radiation: alpha particles, beta particles, and gamma rays.

Alpha particles are positively charged particles, each with a mass approximately four times that of a hydrogen atom. These particles are helium atoms stripped of their orbiting electrons. Beta particles are actually electrons, and gamma rays are electromagnetic radiations similar to X-rays but of a higher frequency and energy (refer to the electromagnetic spectrum in Figure 9–7). Fortunately, most naturally occurring isotopes are not radioactive, and those that are—radium, uranium, and thorium—are found in such small quantities in the earth’s crust that their radioactivity presents no hazard to human survival.

Because of their large mass, alpha particles do not tend to travel far and are not very penetrat- ing; a sheet of paper or your skin easily stops them. However, isotopes that emit alpha particles are dangerous when ingested. The radioisotope polonium-210, an emitter of alpha particles, was implicated in the murder of an ex-KGB agent, as discussed in the following case file.

The existence of isotopes would be of little importance to the forensic chemist were it not for the fact that scientists have mastered the techniques for synthesizing radioactive isotopes. If the only distinction between isotopes of an element is the number of neutrons each possesses, is it not reasonable to assume that when atoms are bombarded with neutrons, some neutrons will be captured to make new isotopes? This is exactly what happens in a nuclear reactor. A nuclear reac- tor is simply a source of neutrons that can be used to bombard the atoms of a specimen, thereby creating radioactive isotopes. When the nucleus of an atom captures a neutron, a new isotope with one additional neutron is formed. In this state, the nuclei are said to be activated, and many immediately begin to decompose by emitting radioactivity.

Neutron Activation Analysis Forensic chemists can characterize the trace elements in a specimen by bombarding it with neu- trons and measuring the energy of the gamma rays emitted by the activated isotopes. The gamma rays of each element can be associated with a characteristic energy value. Furthermore, once the element has been identified, its concentration can be measured by the intensity of its gamma-ray radiation; intensity is directly proportional to the concentration of the element in a specimen. The technique of bombarding specimens with neutrons and measuring the resultant gamma-ray radioactivity is known as neutron activation analysis. The process is depicted in Figure 13–8.

The major advantage of neutron activation analysis is that it provides a nondestructive method for identifying and quantitating trace elements. A median detection sensitivity of one- billionth of a gram (one nanogram) makes neutron activation analysis one of the most sensitive methods available for the quantitative detection of many elements. Further, neutron activation can simultaneously analyze 20 to 30 elements. A major drawback to the technique is its ex- pense and regulatory requirements. Only a handful of crime laboratories worldwide have access to a nuclear reactor; in addition, sophisticated analyzers are needed to detect and discriminate gamma-ray emissions.

As far as forensic analysis is concerned, neutron activation has been used to characterize trace elements present in metals, drugs, paint, soil, gunpowder residues, and hair. A typical illustration

Hydrogen Deuterium Tritium

1P 1P 1n

1P 2n

radioactivity The particle and/or gamma-ray radiation emitted by the unstable nucleus of some isotopes.

alpha particle A type of radiation emitted by a ra- dioactive element; the radiation is composed of helium atoms minus their orbiting electrons.

beta particle A type of radiation emitted by a radioactive element; the radiation consists of electrons.

gamma ray A high-energy form of electromag- netic radiation emitted by a radio- active element.

FIGURE 13–7 Isotopes of hydrogen.

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Death by Radiation Poisoning In November 2006, Alexander V. Litvinenko lay at death’s door in a London hospital. He was in excruciating pain and had symptoms that included hair loss, the inability to make blood cells, and gastrointestinal distress. His organs slowly failed as he lingered for three weeks and then died. British investiga- tors soon confirmed that Litvinenko died from the intake of polonium-210, a radioactive element, in what appeared to be its first use as a murder weapon (see the figure).

Litvinenko’s death almost immediately set off an interna- tional uproar. Litvinenko, a former KGB operative, had become a vocal critic of the Russian spy agency FSB, the domestic suc- cessor to the KGB. In 2000, he fled to London, where he was granted asylum. Litvinenko continued to voice his criticisms of the Russian spy agency and also became highly critical of Russia’s president, Vladimir Putin. Just before his death, he was believed to have compiled, on behalf of a British company looking to invest millions in a project in Russia, an incriminat- ing report regarding the activities of senior Kremlin officials.

Suspicions immediately fell onto Andrei Lugovoi and Dmitri Kovtun, business associates of Litvinenko. Lugovoi was himself a former KGB officer. On the day he fell ill, Litvinenko met Lugovoi and Kovtun at the Pine Bar of the Millennium Hotel in London. At the meeting, Litvinenko drank tea out of a teapot later found to be highly radioactive. British officials have accused Lugovoi of poisoning Litvinenko. The precise

nature of the evidence against him has not been made clear, though investigators have linked him and Kovtun to a trail of polonium-210 radioactivity stretching from hotel rooms, res- taurants, bars, and offices in London to Hamburg, Germany, and to British Airways planes that had flown to Moscow. Each man has denied killing Litvinenko.

Polonium-210 is highly radioactive and very toxic. By weight, it is about 250 million times as toxic as cyanide, so a particle the size of a dust particle could be fatal. It emits a radioactive ray known as an alpha particle. This form of radiation cannot penetrate the skin, so polonium-210 is effective as a poison only if it is swallowed, breathed in, or injected. The particles disperse through the body and first destroy fast-growing cells, like those in bone marrow, blood, hair, and the digestive tract. That would be consistent with Litvinenko’s symptoms. There is no antidote for polonium poisoning.

Polonium does have industrial uses and is produced by commercial or institutional nuclear reactors. Polonium-210 has been found to be ideal for making antistatic devices that remove dust from film and lenses as well as paper and tex- tile plants. Its non-body-penetrating rays produce an electric charge on nearby air. Bits of dust with static attract the charged air, which neutralizes them. Once free of static, the dust is easy to blow or brush away. Manufacturers of such antistatic devices take great pains to make the polonium hard to remove from their products.

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Alexander Litvinenko, former KGB agent, before (right) and after he became sick (left).

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of its application occurred during the investigation of a theft of copper telegraphic wires in Canada. Four lengths of copper wire (A1, A2, A3, A4) found at the scene of the theft were com- pared by neutron activation with a length of copper wire (B) seized at a scrap yard and suspected of being stolen. All were bare, single-strand wire with the same general physical appearance and a diameter of 0.28 centimeter. Prior experiments had revealed that significant variations could be expected in the concentration levels of the trace elements selenium, gold, antimony, and silver IS

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Atom

Neutron

Neutrons bombard specimen

Atom

Neutron

Energy

In te

ns ity

Energy

In te

ns ity

Energy

In te

ns ity

Detector measures the energies and intensities of the gamma rays

Multichannel analyzer

Each element is associated with a characteristic energy value. Intensity indicates the element concentration in the specimen

Atom

Gamma Rays

FIGURE 13–8 The neutron activation process requires the capture of a neutron by the nucleus of an atom. The new atom is now radioactive and emits gamma rays. A detector permits identification of the radioactive atoms present by measuring the energies and intensities of the gamma rays emitted.

for wires originating from different sources. A comparison of these elements present in the wire involved in the theft was undertaken. After exposing the wires to neutrons in a nuclear reactor, neutron activation analysis revealed a match between A1 and B that was well within experimental error (see Table 13–2). The findings suggested a common origin of the control and suspect wires.

Forensic Examination of Paint Our environment contains millions of objects whose surfaces are painted. Thus, it is not surpris- ing to observe that paint, in one form or another, is one of the most prevalent types of physical evidence received by the crime laboratory. Paint as physical evidence is perhaps most frequently

TABLE 13–2 Concentration of Trace Elements in Copper Wire

Selenium Gold Antimony Silver Control Wire A1 2.4 0.047 0.16 12.7 A2 3.5 0.064 0.27 17.2 A3 2.6 0.050 0.20 13.3 A4 1.9 0.034 0.21 12.6 Suspect Wire B 2.3 0.042 0.15 13.0

Note: Average concentration measured in parts per million.

Source: R. K. H. Chan, “Identification of Single-Stranded Copper Wires by Nondestructive Neutron Activation Analysis,” Journal of Forensic Sciences 17 (1972): 93. Reprinted by permission of the American Society for Testing and Materials, copyright 1972.

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Nuclear forensics can be performed on a broad spectrum of substances. An example is stolen contain- ers of uranium diverted during one of the mining, mill- ing, conversion, enrichment, or fuel fabrication steps used to convert uranium ore to enriched fuel for nuclear power plants; uranium varies in isotopic composition and impurities according to where the uranium was mined and how it was processed. Another example is commercial radioactive materials used in applications such as medical diagnostics and food sterilization.

Researchers analyze the material’s chemical and isotopic composition, which includes measuring the amounts of trace elements as well as the ratio of parent isotopes to daughter isotopes. These measurements help determine the source location and sample’s age. They also examine the material’s morphological charac- teristics such as shape, size, and texture. Analytical meth- ods include electron microscopy, X-ray diffraction, and mass spectrometry. In addition, as a sample is moved from place to place, it picks up trace evidence such as pollen, hairs, fibers, plant DNA, and fingerprints. These so-called route materials may provide information about who has handled a sample and the path it has traveled.

When comparing a sample’s signature against known signatures from uranium mines and fabrication plants, researchers can benefit by assembling a library of nuclear materials of known origin from around the world. Nuclear scientists have developed relationships with domestic suppliers of nuclear materials to assem- ble such a library. Contracts with major U.S. uranium fuel suppliers have provided researchers with sam- ples and manufacturing data. Forensic scientists are also seeking to obtain samples of uranium products worldwide to analyze the products’ isotopic and trace- element content, grain size, and microstructure. Na- tions with nuclear capabilities are beginning to share information about their nuclear fuel processes and ma- terials. The development of databases is essential to the nuclear forensic scientist’s mission of identifying the origin of nuclear materials intercepted in the black market or associated with a terrorist event.

Nuclear Forensics

Nuclear forensics has emerged as a critical profession on the forefront in the war on terrorism. Nuclear foren- sic scientists are responsible for developing ways to analyze nuclear materials recovered from either inter- cepted intact nuclear materials or postexplosion de- bris created as a result of a nuclear explosion. Nuclear forensics can trace its origin to the cold war era, when U.S. planes surreptitiously flew over Soviet airspace sampling airborne particles from the country’s nuclear bomb tests. Nuclear forensics matured as a science when the Soviet empire disintegrated and concerns arose over the security of nuclear materials located in states of the former Soviet Union. Fears that these materials might fall into the hands of terrorist organiza- tions engendered scenarios of dirty nuclear bomb at- tacks on the United States and other Western nations.

Nuclear forensics is becoming an increasingly im- portant tool in the fight against illegal smuggling and trafficking of radiological and nuclear materials. These include materials intended for industrial and medical use, nuclear materials such as those produced in the nuclear fuel cycle of a nuclear power plant (see the figure), and much more dangerous nuclear materials that can be used in weapons, such as plutonium and highly enriched uranium. Since the early 1990s, more than two hundred cases of illicitly trafficked nuclear materials have been reported.

In the United States, Lawrence Livermore National Laboratory along with seven other Department of En- ergy (DOE) national laboratories have been tasked by the FBI and the Department of Homeland Secu- rity with developing the nation’s technical forensics capability for nuclear and radiological materials. Or- ganizations such as the European Commission’s Insti- tute for Transuranium Elements, located in Karlsruhe, Germany, have extended nuclear forensic capabilities onto an international scale.

A major focus of nuclear forensics is identifying signatures, which are the physical, chemical, and iso- topic characteristics that distinguish one nuclear or radiological material from another. Signatures enable researchers to identify the processes used to initially create a material, which ultimately may yield clues as to the origin of the seized material.

Attribution is the integration of all information, including forensic data, law enforcement and intelli- gence data, to corroborate or exclude the origin of nuclear materials and devices, routes of transit and responsible groups or individuals.

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View of a nuclear power plant.

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encountered in hit-and-run and burglary cases. For example, a chip of dried paint or a paint smear may be transferred to the clothing of a hit-and-run victim on impact with an automobile, or paint smears could be transferred onto a tool during the commission of a burglary. Obviously, in many situations a transfer of paint from one surface to another could impart an object with an identifi- able forensic characteristic.

In most circumstances, the criminalist must compare two or more paints to establish their common origin. For example, such a comparison may associate an individual or a vehicle with the crime site. However, the criminalist need not be confined to comparisons alone. Crime labo- ratories often help identify the color, make, and model of an automobile by examining small quantities of paint recovered at an accident scene. Such requests, normally made in connection with hit-and-run cases, can lead to the apprehension of the responsible vehicle.

Composition of Paint Paint spread onto a surface dries into a hard film consisting of pigments and additives suspended in a binder. Pigments impart color and hiding (or opacity) to paint and are usually mixtures of different inorganic and organic compounds added to the paint by the manufacturer to produce specific colors and properties. The binder provides the support medium for the pigments and ad- ditives and is a polymeric substance. Paint is thus composed of a binder and pigments, as well as other additives, all dissolved or dispersed in a suitable solvent. After the paint has been applied to a surface, the solvent evaporates, leaving behind a hard polymeric binder and any pigments that were suspended in it.

One of the most common types of paint examined in the crime laboratory is finishes from au- tomobiles. One interesting fact that is helpful in forensic characterization of automotive paint is that manufacturers apply a variety of coatings to the body of an automobile. This adds significant diver- sity to automobile paint and contributes to the forensic significance of automobile paint compari- sons. The automotive finishing system for steel usually consists of at least four organic coatings:

Electrocoat Primer The first layer applied to the steel body of a car is the electrocoat primer. The primer, consisting of epoxy-based resins, is electroplated onto the steel body of the automobile to provide corrosion resistance. The resulting coating is uniform in appearance and thickness. The color of these electrodeposition primers ranges from black to gray.

Primer Surfacer Originally responsible for corrosion control, the surfacer usually follows the electrocoat layer and is applied before the basecoat. Primer surfacers are epoxy-modified polyesters or urethanes. The function of this layer is to completely smooth out and hide any seams or imperfections because the colorcoat will be applied on this surface. This layer is highly pigmented. Color pigments are used to minimize color contrast between primer and topcoats. For example, a light-gray primer may be used under pastel shades of a colored topcoat; a red oxide may be used under a dark-colored topcoat.

Basecoat The next layer of paint on a car is the basecoat or colorcoat. This layer provides the color and aesthetics of the finish and represents the “eye appeal” of the finished automobile. The integrity of this layer depends on its ability to resist weather, UV radiation, and acid rain. Most commonly, an acrylic-based polymer comprises the binder system of basecoats. Interestingly, the choice of automotive pigments is dictated by toxic and environmental concerns. Thus, the use of lead, chrome, and other heavy-metal pigments has been abandoned in favor of organic-based pigments. There is also a growing trend toward pearl luster or mica pigments. Mica pigments are coated with layers of metal oxide to generate interference colors. Also, the addition of aluminum flakes to automotive paint imparts a metallic look to the paint’s finish.

Clearcoat An unpigmented clearcoat is applied to improve gloss, durability, and appearance. Most clearcoats are acrylic based, but polyurethane clearcoats are increasing in popularity. These topcoats provide outstanding etch resistance and appearance.

Microscopic Examination of Paint The microscope has traditionally been and remains the most important instrument for locat- ing and comparing paint specimens. Considering the thousands of paint colors and shades, it is quite understandable why color, more than any other property, imparts paint with its most

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distinctive forensic characteristics. Questioned and known specimens are best compared side by side under a stereoscopic microscope for color, surface texture, and color layer sequence (see Figure 13–9).

The importance of layer structure for evaluating the evidential significance of paint evidence cannot be overemphasized. When paint specimens possess colored layers that match in number and sequence of colors, the examiner can begin to relate the paints to a common origin. How many layers must be matched before the criminalist can conclude that the paints come from the same source? There is no one accepted criterion. Much depends on the uniqueness of each layer’s color and texture, as well as the frequency with which the particular combination of colors under inves- tigation is observed to occur. Because no books or journals have compiled this type of information, the criminalist is left to his or her own experience and knowledge when making this decision.

Unfortunately, most paint specimens presented to the criminalist do not have a layer structure of sufficient complexity to allow them to be individualized to a single source (see Figure 13–10). However, the diverse chemical composition of modern paints provides additional points of

FIGURE 13–10 Red paint chips peeling off a wall revealing underlying layers.

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FIGURE 13–9 A stereoscopic microscope comparison of two automotive paints. The questioned paint on the left has a layer structure consistent with P the control paint on the right.

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comparison between specimens. Specifically, a thorough comparison of paint must include a chemical analysis of the paint’s pigments, its binder composition, or both.

Analytical Techniques Used in Paint Comparison The wide variation in binder formulations in automobile finishes provides particularly significant information. More important, paint manufacturers make automobile finishes in hundreds of variet- ies; this knowledge is most helpful to the criminalist who is trying to associate a paint chip with one car as distinguished from the thousands of similar models that have been produced in any one year. For instance, there are more than a hundred automobile production plants in the United States and Canada. Each can use one paint supplier for a particular color or vary suppliers during a model year. Although a paint supplier must maintain strict quality control over a paint’s color, the batch formulation of any paint binder can vary, depending on the availability and cost of basic ingredients.

CHARACTERIZATION OF PAINT BINDERS Pyrolysis gas chromatography has proven to be a particularly invaluable technique for distinguishing most paint formulations. In this process, paint chips as small as 20 micrograms are decomposed by heat into numerous gaseous products and are sent through a gas chromatograph. As shown in Figure 13–11, the polymer chain is decomposed by a heated filament, and the resultant products are swept into and through a gas chromatograph column. The separated decomposition products of the polymer emerge and are recorded. The pattern of this chromatogram or “pyrogram” distinguishes one polymer from another. The result is a pyrogram that is sufficiently detailed to reflect the chemical makeup of the binder. Figure 13–12 illustrates how the patterns produced by paint pyrograms can differentiate acrylic enamel paints removed from two different automobiles. Infrared spectrophotometry is still another analytical technique that provides information about the binder composition of paint.2 Binders selectively absorb infrared radiation to yield a spectrum that is highly characteristic of a paint specimen.

CHARACTERIZATION OF PIGMENTS The elements that constitute the inorganic pigments of paints can be identified by a variety of techniques—emission spectroscopy, inductively coupled plasma (ICP), and X-ray spectroscopy (page 162). The emission spectrograph, for instance, can simultaneously detect 15 to 20 elements in most automobile paints. Some of these elements are relatively common to all paints and have little forensic value; others are less frequently encountered and provide excellent points of comparison between paint specimens.

2 P. G. Rodgers et al., “The Classification of Automobile Paint by Diamond Window Infrared Spectrophotometry, Part I: Binders and Pigments,” Canadian Society of Forensic Science Journal 9 (1976): 1; T. J. Allen, “Paint Sample Presentation for Fourier Transform Infrared Microscopy,” Vibration Spectroscopy 3 (1992): 217.

pyrolysis The decomposition of organic matter by heat.

Carrier gas

Pyrolyzer

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FIGURE 13–11 Schematic diagram of pyrolysis gas chromatography.

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Significance of Paint Evidence Once a paint comparison is completed, the task of assessing the significance of the finding be- gins. How certain can one be that two similar paints came from the same surface? For instance, a casual observer sees countless identically colored automobiles on our roads and streets. If this is the case, what value is a comparison of a paint chip from a hit-and-run scene to paint removed from a suspect car? From previous discussions it should be apparent that far more is involved in paint comparison than matching surface paint colors. Paint layers present beneath a surface layer offer valuable points of comparison. Furthermore, forensic analysts can detect subtle differences in paint binder formulations, as well as major or minor differences in the elemental composition of paint. Obviously, these properties cannot be discerned by the naked eye.

The significance of a paint comparison was convincingly demonstrated from data gath- ered at the Centre of Forensic Sciences, Toronto, Canada.3 Paint chips randomly taken from 260 vehicles located in a local wreck yard were compared by color, layer structure, and, when required, by infrared spectroscopy. All were distinguishable except for one pair. In statistical terms, these results signify that if a crime-scene paint sample and a paint standard/reference sample removed from a suspect car compare by the previously discussed tests, the odds against the crime-scene paint originating from another randomly chosen vehicle are approximately 33,000 to 1. Obviously, this type of evidence is bound to forge a strong link between the suspect car and the crime scene.

After automotive paints, architectural paint comparisons are the most common paint analyses required of forensic laboratories. A large-scale population study of architectural paints collected throughout North America has been conducted for their forensic value.4 Inter- comparisons of nearly 960 randomly collected paints by visual, microscopic, and infrared technology resulted in a 99.99 percent differentiation of the paints, thus demonstrating the high diversity of architectural paints in our environment and their meaningful forensic value as class evidence.

Time (minutes) 2 4 6 8 10 12

(a) Time (minutes)

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FIGURE 13–12 Paint pyrograms of acrylic enamel paints. (a) Paint from a Ford model and (b) paint from a Chrysler model.

3 G. Edmondstone, J. Hellman, K. Legate, G. L. Vardy, and E. Lindsay, “An Assessment of the Evidential Value of Automotive Paint Comparisons,” Canadian Society of Forensic Science Journal 37 (2004): 147.

4 D. W. Wright et al., “Analysis and Documentation of Architectural Paint Samples via a Population Study,” Forensic Science International 209 (2011): 86.

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Crime laboratories are often asked to identify the make and model of a car from a very small amount of paint left behind at a crime scene. Such information is frequently of use in a search for an unknown car involved in a hit-and-run incident. Often, the questioned paint can be identified when its color is compared to color chips representing the various makes and models of manufactured cars. However, in many cases it is not possible to state the exact make or model of the car in question be- cause any one paint color can be found on more than one car model. For example, General Motors may use the same paint color for several production years on cars in its Cadillac, Buick, and Chevrolet lines.

Color charts for automobile finishes are available from various paint manufacturers and refinishers (see Figure 13–13). Starting with the 1974 model year, the Law Enforcement Stan- dards Laboratory at the National Institute of Standards and Technology collected and dissemi- nated to crime laboratories auto paint color samples from U.S. domestic passenger cars. This collection was distributed by Collaborative Testing Services, McLean, Virginia, through 1991. Since 1975, the Royal Canadian Mounted Police Forensic Laboratories have been systemati- cally gathering color and chemical information on automotive paints. This computerized data- base, known as PDQ (Paint Data Query), allows an analyst to obtain information on paints related to automobile make, model, and year. The database contains such parameters as automo- tive paint layer colors, primer colors, and binder composition. A number of U.S. laboratories have access to PDQ.5 Also, some laboratories maintain an in-house collection of automobile paints associated with various makes and models.

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FIGURE 13–13 Automotive color chart of various car models.

5 J. L. Buckle et al., “PDQ—Paint Data Queries: The History and Technology behind the Development of the Royal Canadian Mounted Police Laboratory Services Automotive Paint Database,” Canadian Society of Forensic Science Journal 30 (1997): 199. An excellent discussion of the PDQ database is also available in A. Beveridge, T. Fung, and D. MacDougall, “Use of Infrared Spectroscopy for the Characterisation of Paint Fragments,” in B. Caddy, ed., Forensic Examination of Glass and Paint (New York: CRC Press, 2001), pp. 222–233.

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Collection and Preservation of Paint Evidence As has already been noted, paint chips are most likely to be found on or near people or objects involved in hit-and-run incidents. The recovery of loose paint chips from a garment or from the road surface must be done with the utmost care to keep the paint chip intact. Paint chips may be picked up with a tweezers or scooped up with a piece of paper. Paper druggist folds and glass or plastic vials make excellent containers for paint. If the paint is smeared on or embedded in gar- ments or objects, the investigator should not attempt to remove it; instead, it is best to package the whole item carefully and send it to the laboratory for examination.

When a transfer of paint occurs in hit-and-run situations, such as to the clothing of a pedes- trian victim, uncontaminated standard/reference paint must always be collected from an undam- aged area of the vehicle for comparison in the laboratory. It is particularly important that the collected paint be close to the area of the car that was suspected of being in contact with the victim. This is necessary because other portions of the car may have faded or been repainted. Standard/reference samples are always removed so as to include all the paint layers down to the bare metal. This is best accomplished by removing a painted section with a clean scalpel or knife blade. Samples ¼ inch square are sufficient for laboratory examination. Each paint sample should be separately packaged and marked with the exact location of its recovery. When a cross- transfer of paint occurs between two vehicles, again all of the layers, including the foreign as well as the underlying original paints, must be removed from each vehicle. A standard/reference sample from an adjacent undamaged area of each vehicle must also be taken in such cases. Care- fully wipe the blade of any knife or scraping tool used before collecting each sample, to avoid cross-contamination of paints.

Tools used to enter buildings or safes often contain traces of paints as well as other sub- stances such as wood and safe insulation. Care must be taken not to lose this type of trace ev- idence. The scene investigator should not try to remove the paint; instead, he or she should package the tool for laboratory examination. Standard/reference paint should be collected from all surfaces suspected of having been in contact with the tool. Again, all layers of paint must be included in the sample.

The Predator September in Arizona is usually hot and dry, much like the rest of the year—but September 1984 was a little different. Unusu- ally heavy rains fell for two days, which must have seemed fitting to the friends and family of 8-year-old Vicki Lynn Hoskinson. Vicki went missing on September 17 of that year, and her disappearance was investigated as a kidnapping. A schoolteacher who knew Vicki remembered seeing a suspicious vehicle loitering near the school that day, and he happened to jot down the license plate number. This crucial tip led police to 28-year-old Frank Atwood, recently paroled from a California prison. Police soon learned that Atwood had been convicted for committing sex offenses and for kidnapping a boy. This gal- vanized the investigators, who realized Vicki could be at the mercy of a dangerous and perverse man.

The only evidence the police had to work with was Vicki’s bike, which was found abandoned in the middle of the street a few blocks from her home. Police found scrapes from her bike pedal on the underside of the gravel pan on Atwood’s car, as well as pink paint on Atwood’s front bumper, apparently transferred from Vicki’s bike. The police believed that Atwood deliberately struck Vicki while she was riding her bicycle, knocking her to the ground.

The pink paint on Atwood’s bumper was first looked at microscopically and then examined by pyrolysis gas

chromatography. This technique provides investigators with a “fingerprint” pattern of the paint sample, enabling them to compare this paint to any other paint evidence. In this case, the pink paint on Atwood’s bumper matched the paint from Vicki’s bicycle.

Vicki’s skeletal remains were discovered in the desert, several miles from her home, in the spring of 1985. Positive identification was made using dental records, but investiga- tors wanted to see if the remains could help them determine how long she had been dead. Atwood had been jailed on an unrelated charge three days after Vicki disappeared, so the approximate date of death was very important to proving his guilt.

Investigators found adipocere, a white, fatty residue produced during decomposition, inside Vicki’s skull. This provided evidence that moisture was present around Vicki’s body after her death, which did not seem to make sense, con- sidering her body was found in the Arizona desert! A check of weather records revealed that there had been an unusual amount of rainfall during only one period of time since Vicki was last seen alive: a mere 48 hours after her disappearance. This put Vicki’s death squarely within Frank Atwood’s three- day window of opportunity between her disappearance and his arrest. Frank Atwood was sentenced to death in 1987 for the murder of Vicki Lynn Hoskinson. He remains on death row awaiting execution.

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When the tool has left its impression on a surface, standard/reference paint is collected from an uncontaminated area adjacent to the impression. No attempt should be made to collect the paint from the impression itself. If this is done, the impression may be permanently altered and its evidential value lost.

Forensic Analysis of Soil There are many definitions for the term soil; however, for forensic purposes, soil may be thought of as any disintegrated material, natural and/or artificial, that lies on or near the earth’s surface. Therefore, forensic examination of soil is not only concerned with the analysis of naturally oc- curring rocks, minerals, vegetation, and animal matter; it also encompasses the detection of such manufactured objects as glass, paint chips, asphalt, brick fragments, and cinders, whose presence may impart soil with characteristics that make it unique to a particular location. When this ma- terial is collected accidentally or deliberately in a manner that associates it with a crime under investigation, it becomes valuable physical evidence.6

Significance of Soil Evidence The value of soil as evidence rests on its prevalence at crime scenes and its transferability be- tween the scene and the criminal. Thus, soil or dried mud found adhering to a suspect’s clothing or shoes or to an automobile, when compared to soil samples collected at the crime site, may link a suspect or object to the crime scene. As with most types of physical evidence, forensic soil analysis is comparative in nature; soil found in the possession of the suspect must be carefully collected and then compared to soil samplings from the crime scene and its vicinity.

However, one should not rule out the value of soil even if the site of the crime has not been as- certained. For instance, small amounts of soil may be found on a person or object far from the actual site of a crime. A geologist who knows the local geology may be able to use geological maps to direct police to the general vicinity where the soil was originally picked up and the crime committed.

Forensic Examination of Soil Most soils can be differentiated by their gross appearance. A side-by-side visual comparison of the color and texture of soil specimens is easy to perform and provides a sensitive property for distinguishing soils that originate from different locations. Soil is darker when it is wet; therefore, color comparisons must always be made when all the samples are dried under identical labora- tory conditions. It is estimated that there are nearly 1,100 distinguishable soil colors; hence, color offers a logical first step in a forensic soil comparison (see Figure 13–14).

Low-power microscopic examination of soil reveals the presence of plant and animal ma- terials as well as artificial debris. Further high-power microscopic examination helps character- ize minerals and rocks in earth materials. Although this approach to forensic soil identification requires the expertise of an investigator trained in geology, it can provide the most varied and significant points of comparison between soil samples. Only by carefully examining and compar- ing the minerals and rocks naturally present in soil can one take advantage of the large number of variations between soils and thus add to the evidential value of a positive comparison.7

A mineral is a naturally occurring crystal, and like any other crystal, its physical properties—for example, its color, geometric shape, density, and refractive index—are useful for identification. More than 2,200 minerals exist; however, most are so rare that forensic geologists usually encounter only about 20 of them. Rocks are composed of a combination of minerals and therefore exist in thousands of varieties on the earth’s surface. They are usually identified by characterizing their mineral content and grain size (see Figure 13–15).

Considering the vast variety of minerals and rocks and the possible presence of artificial debris in soil, the forensic geologist is presented with many points of comparison between two or more specimens. The number of comparative points and their frequency of occurrence must

6 E. P. Junger, “Assessing the Unique Characteristics of Close-Proximity Soil Samples: Just How Useful Is Soil Evidence?” Journal of Forensic Sciences 41 (1996): 27.

7 W. J. Graves, “A Mineralogical Soil Classification Technique for the Forensic Scientist,” Journal of Forensic Sciences 24 (1979): 323; M. J. McVicar and W. J. Graves, “The Forensic Comparison of Soil by Automated Scanning Electron Microscopy,” Canadian Society of Forensic Science Journal 30 (1997): 241.

mineral A naturally occurring crystalline solid.

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FIGURE 13–14 A technician classifies a soil sample based on a soil color chart.

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Rocks and minerals not only are present in earth materials but also are used to manufacture a wide variety of industrial and commercial products. For example, the tools and garments of an individual suspected of breaking into a safe often contain traces of safe insulation. Safe insula- tion may be made from a wide combination of mineral mixtures that provide significant points of identification. Similarly, building materials such as brick, plaster, and concrete blocks are IS

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combinations of minerals and rocks that can easily be recognized and compared microscopically to similar minerals found on the breaking-and-entering suspect.

Variations in Soil The ultimate forensic value of soil evidence depends on its uniqueness at the crime scene. If, for example, soil composition is indistinguishable for miles surrounding the location of a crime, as- sociating soil found on the suspect with that particular site will have limited value. Significant conclusions that link a suspect with a particular location through a soil comparison may be made when variations in soil composition occur every 10 to 100 yards from the crime site. However, even when such variations do exist, the forensic geologist usually cannot individualize soil to any one location unless it contains an unusual combination of rare minerals, rocks, or artificial debris.

No statistically valid forensic studies have examined the variability of soil evidence. A study conducted in southern Ontario, Canada, seems to indicate that soil in that part of Canada shows extensive diversity. It estimates a probability of less than 1 in 50 of finding two soils that are indis- tinguishable in both color and mineral properties but originate in two different locations separated by a distance of at least 1,000 feet. Based on these preliminary results, similar diversity may be ex- pected in the northern United States, Canada, northern Europe, and eastern Europe. However, such probability values can only generally indicate the variation of soil within these geographical areas. Each crime scene must be evaluated separately to establish its own soil variation probabilities.

Collection and Preservation of Soil Evidence When gathering soil specimens, the evidence collector must give primary consideration to es- tablishing the variation of soil at the crime-scene area. For this reason, standard/reference soils should be collected at various intervals within a 100 foot radius of the crime scene, as well as at the site of the crime, for comparison to the questioned soil. Soil specimens also should be collected at all possible alibi locations that the suspect may have claimed.

All specimens gathered should be representative of the soil that was removed by the suspect. In most cases, only the top layer of soil is picked up during the commission of a crime. Thus,

Soil: The Silent Witness Alice Redmond was reported missing by her husband on a Monday night in 1983. Police learned that she had been seen with a co-worker, Mark Miller, after work that evening. When police questioned Miller, he stated that the two just “drove around” after work and then she dropped him off at home. De- spite his statement, Miller was the prime suspect because he had a criminal record for burglary and theft.

Alice’s car was recovered in town the following morning. The wheel wells were thickly coated in mud, which investi- gators hoped might provide a good lead. These hopes were dampened when police learned that Alice and her husband had attended a motorcycle race on Sunday, where her car was driven through deep mud.

After careful scrutiny, analysts found two colors of soil on the undercarriage of Alice’s car. The thickest soil was brown; on top of the brown layer was a reddish soil that looked unlike any- thing in the county. Investigators hoped the reddish soil, which had to have been deposited sometime after the Sunday night mo- torcycle event and before the vehicle was discovered on Tuesday morning, could link the vehicle to the location of Alice Redmond.

An interview with Mark Miller’s sister provided a break in the case. She told police that Mark had visited her

on Monday evening. During that visit, he confessed that he had driven Alice in her car across the Alabama state line into Georgia, killed her, and buried her in a remote location. Now that investigators had a better idea where to look for Alice, fo- rensic analysts took soil samples that would prove or disprove Miller’s sister’s story.

Each field sample was dried and compared for color and texture by eye and stereomicroscopy to the reddish-colored soil gathered from the car. Next, soils that compared to the car were passed through a series of mesh filters, each of a finer gauge than the last. In this way, the components of the soil samples were physically separated by size. Finally, each fraction was analyzed and compared for mineral composition with the aid of a polarizing light microscope.

Only samples collected from areas across the Alabama state line near the suspected dump site were consistent with the topmost reddish soil recovered from Alice’s car. This finding supported Miller’s sister’s story and was instrumen- tal in Mark Miller’s being charged with murder and kidnap- ping. After pleading guilty, the defendant led the authorities to where he had buried the body. The burial site was within a half mile of the location where forensic analysts had col- lected a soil sample consistent with the soil removed from Alice’s vehicle.

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standard/reference specimens must be removed from the surface, without digging into the un- representative subsurface layers. Approximately a tablespoon or two of soil in each sample is all the laboratory needs for a thorough comparative analysis. All specimens collected should be packaged in individual containers, such as plastic vials. Each vial should be marked to indicate the location at which the sampling was made.

Soil found on a suspect must be carefully preserved for analysis. If it is found adhering to an object, as in the case of soil on a shoe, the investigator must not remove it. Instead, each object should be individually wrapped in paper, with the soil intact, and transmitted to the laboratory. Similarly, loose soil adhering to garments should not be removed; these items should be carefully and individually wrapped in paper bags and sent to the laboratory for analysis. Care must be taken that particles that may fall off the garment during transportation will remain in the paper bag.

When a lump of soil is found, it should be collected and preserved intact. For example, an automobile tends to collect and build up layers of soil under the fenders, body, and so on. The impact of an automobile with another object may jar some of this soil loose. Once the suspect car has been apprehended, a comparison of the soil left at the scene with soil remaining on the automobile may help establish that the car was present at the accident scene. In these situations, separate samples are collected from under all the fender and frame areas of the vehicle; care is taken to remove the soil in clump form to preserve the order in which the particles of soil adhered to the car and to the other soil on the car. Undoubtedly, during the normal use of an automobile, soil will be picked up from numerous locations over a period of months and years. This layer- ing effect may impart soil with greater variation, and hence greater evidential value, than that normally associated with loose soil.

chapter summary Many manufactured products and even most natural materials contain small quantities of elements in concentrations of less than 1 percent. For the criminalist, the presence of these trace elements is particularly useful because they provide “invis- ible” markers that may establish the source of a material or at least provide additional points for comparison.

Emission spectroscopy and inductively coupled plasma are techniques available to forensic scientists for determining the elemental composition of materials. An emission spec- trograph vaporizes and heats samples to a high temperature so that the atoms present in the material achieve an “excited” state. Under these circumstances, the excited atoms emit light. If the light is separated into its components, one observes a line spectrum. Each element present in the spectrum can be identified by its characteristic line frequencies. In inductively coupled plasma, the sample, in the form of an aerosol, is intro- duced into a hot plasma, creating charged particles that emit light of characteristic wavelengths corresponding to the iden- tity of the elements present.

Neutron activation analysis measures the gamma-ray frequencies of specimens that have been bombarded with neutrons. This method provides a highly sensitive and nonde- structive analysis for simultaneously identifying and quanti- tating 20 to 30 trace elements. Because this technique requires

access to a nuclear reactor, however, it has limited value to forensic analysis.

Paint spread onto a surface dries into a hard film consist- ing of pigments and additives suspended in the binder. One of the most common types of paint examined in the crime labora- tory is finishes from automobiles. Automobile manufacturers normally apply a variety of coatings to the body of an automo- bile. Hence, the wide diversity of automotive paint contributes to the forensic significance of an automobile paint compari- son. Questioned and known specimens are best compared side by side under a stereoscopic microscope for color, surface tex- ture, and color layer sequence. Pyrolysis gas chromatography and infrared spectrophotometry are invaluable techniques for distinguishing most paint binder formulations, adding further significance to a forensic paint comparison.

The value of soil as evidence rests with its prevalence at crime scenes and its transferability between the scene and the criminal. Most soils can be differentiated by their gross appearance. A side-by-side visual comparison of the color and texture of soil specimens is easy to perform and pro- vides a sensitive property for distinguishing soils that origi- nate from different locations. In many forensic laboratories, forensic geo logists characterize and compare the mineral content of soils.

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review questions

1. The presence of ___________ elements in materials provides useful “invisible” markers when comparing physical evidence.

2. The proton and electron (are, are not) of approximately equal mass.

3. A proton imparts the nucleus of an atom with a ___________ charge.

4. The number of protons (is, is not) always equal to the number of electrons in orbit around the nucleus of an atom.

5. Each atom of the same element always has the same number of ___________ in its nucleus.

6. The number of protons in the nucleus of an atom is called the ___________.

7. The knowledge that elements selectively ___________ and ___________ light provides the basis for important analytical techniques designed to detect the presence of elements in materials.

8. A(n) ___________ is a display of colors or frequencies emitted from a light source.

9. True or False: A continuous spectrum consists of a blending of colors. ___________

10. A(n) ___________ spectrum shows distinct frequencies or wavelengths of light.

11. A line spectrum of an element (is, is not) characteristic of the element.

12. Three important subatomic particles of the atom are the ___________, ___________, and ___________.

13. The total number of protons and neutrons present in a nucleus is known as the ___________.

14. Atoms differing only in the number of neutrons present in their nuclei are called ___________.

15. True or False: Deuterium has the greatest number of protons of all the isotopes of hydrogen. ___________

16. Radioactivity is composed of the following emissions: ___________, ___________, and ___________.

17. Beta particles are identical to ___________.

18. Electromagnetic waves similar to X-rays but of a higher energy are ___________.

19. A nuclear reactor is a source of ___________.

20. The technique of bombarding specimens with neutrons and measuring the resultant gamma ray emissions is known as ___________.

21. The two most important components of dried paint from the criminalist’s point of view are the ___________ and the ___________.

22. The most important physical property of paint in a fo- rensic comparison is ___________.

23. Paints can be individualized to a single source only when they have a sufficiently detailed ___________.

24. The ___________ layer provides corrosion resistance for the automobile.

25. “Eye appeal” of the automobile comes from the ___________ layer.

26. Pyrolysis gas chromatography is a particularly valu- able technique for characterizing paint’s (binder, pigments).

27. True or False: Emission spectroscopy can be used to identify the components of paint’s pigments.

28. True or False: Paint samples removed for examination must always include all of the paint layers. ___________

29. True or False: Most soils have indistinguishable color and texture. ___________

30. Naturally occurring crystals commonly found in soils are ___________.

31. True or False: The ultimate value of soil as evidence de- pends on its variation at the crime scene. ___________

32. To develop an idea of the soil variation within the crime- scene area, standard/reference soils should be collected at various intervals within a(n) ___________ foot radius of the crime scene.

33. True or False: Each object collected at the crime scene that contains soil evidence must be individually wrapped in plastic, with the soil intact, and transmitted to the laboratory.

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review questions for inside the science

1. True or False: Matter in a solid or liquid state pro- duces an emission spectrum that is characteristic of its composition. ___________

2. The ___________ is an instrument used to obtain and record the line spectrum of elements.

3. Excitation of a specimen can be accomplished when it is inserted between two ___________ electrodes.

4. True or False: Each element has its own characteristic set of energy levels. ___________

5. True or False: To move an electron from one energy level to the next requires a definite amount of energy. ___________

6. As an electron falls from a higher to a lower energy level, it emits ___________.

1. You are investigating a hit-and-run accident and have identified a suspect vehicle. Describe how you would collect paint to determine whether the suspect vehicle was involved in the accident. Be sure to indicate the tools you would use and the steps you would take to pre- vent cross-contamination.

2. A forensic analyst at the local crime lab receives pieces of a disfigured bullet from a crime scene. She then ob- tains an exemplar bullet fired by the firearms analyst from the suspect’s firearm. What is the next step in analysis?

3. Only a handful of crime laboratories worldwide have access to a nuclear reactor to carry out neutron activa- tion analysis. What are some possible reasons why this is so?

4. Criminalist Jared Heath responds to the scene of an assault, on an unpaved lane in a rural neighborhood. Rain had fallen steadily the night before, making the area quite muddy. A suspect with very muddy shoes was apprehended nearby but claims to have picked up the mud either from his garden or from the unpaved parking lot of a local restaurant. Jared uses a spade to remove several samples of soil, each about 2 inches deep, from the immediate crime scene and places each in a sepa- rate plastic vial. He collects the muddy shoes and wraps them in plastic as well. At the laboratory, he unpack- ages the soil samples and examines them carefully, one at a time. He then analyzes the soil on the shoes to see whether it matches the soil from the crime scene. What mistakes, if any, did Jared make in his investigation?

application and critical thinking

further references

Caddy, B., ed., Forensic Examination of Glass and Paint. Boca Raton, Fla.: CRC Press, 2001.

Forensic Analysis: Weighing Bullet Lead Evidence. Washington, D.C.: National Academies Press, 2004.

Houck, Max M., ed., Mute Witnesses: Trace Evidence Analysis. Burlington, Mass.: Elsevier Academic Press, 2001.

Houck, Max M., ed., Trace Evidence Analysis—More Cases in Mute Witnesses. Burlington, Mass.: Elsevier Academic Press, 2004.

Murray, R. C., Evidence from the Earth: Forensic Geology and Criminal Investigation. Missoula, Mont.: Mountain Press, 2004.

Murray, R. C., and L. P. Solebello, “Forensic Examination of Soil,” in R. Saferstein, ed., Forensic Science Handbook, vol. 1, 2nd ed. Upper Saddle River, N.J.: Prentice Hall, 2002.

Pye, K., Geological and Soil Evidence: Forensic Applications. Baca Raton, Fla.: CRC Press, 2007.

Thornton, J. L., “Forensic Paint Examination,” in R. Saferstein, ed., Forensic Science Handbook, vol. 1, 2nd ed. Upper Saddle River, N.J.: Prentice Hall, 2002.

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O. J. Simpson—A Mountain of Evidence

On June 12, 1994, police arrived at the home of Nicole Simpson only to view a horrific scene. The bodies of O. J. Simpson’s estranged wife and her friend Ron Goldman were found on the path leading to the front door of Nicole’s home. Both bodies were covered in blood and had suffered deep knife wounds. Nicole’s head was nearly severed from her body. This was not a well-planned murder. A trail of blood led away from the murder scene. Blood was found in O. J. Simpson’s Bronco. Blood drops were on O. J.’s driveway and in the foyer of his home. A blood-soaked sock was located in O. J. Simpson’s bedroom, and a bloodstained glove rested outside his residence.

As DNA was extracted and profiled from each bloodstained article, a picture emerged that seemed to

irrefutably link Simpson to the murders. A trail of DNA leaving the crime scene was consistent with O. J.’s profile, as was the DNA found entering Simpson’s

home. Simpson’s DNA profile was found in the Bronco along with that of both victims. The glove contained the DNA profiles of Nicole and Ron, and the sock had Nicole’s DNA profile. At trial, the defense team valiantly fought back. Miscues in evidence collection were craftily exploited. The defense strategy was to paint a picture of not only an incompetent investigation, but one that was tinged with dishonest police planting evidence. The strategy worked. O. J. Simpson was acquitted of murder.

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After studying this chapter you should be able to: List the A-B-O antigens and antibodies found in the blood for each of the four blood types: A, B, AB, and O

Understand and describe how whole blood is typed

List and describe forensic tests used to characterize a stain as blood

Understand the concept of antigen–antibody interactions and how it is applied to species identification and drug identification

Explain the differences between monoclonal and polyclonal antibodies

Contrast chromosomes and genes

Learn how the Punnett square is used to determine the geno- types and phenotypes of offspring

List the laboratory tests necessary to characterize seminal stains

Explain how suspect blood and semen stains are to be prop- erly preserved for laboratory examination

Describe the proper collection of physical evidence in a rape investigation

forensic serology

acid phosphatase agglutination allele antibody antigen antiserum aspermia chromosome deoxyribonucleic acid

(DNA) egg erythrocyte gene genotype hemoglobin heterozygous homozygous hybridoma cells locus luminol monoclonal antibodies oligospermia phenotype plasma polyclonal antibodies precipitin serology serum sperm X chromosome Y chromosome zygote

KEY TERMS

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354 CHAPTER 14

In 1901, Karl Landsteiner announced one of the most significant discoveries of the 20th century—the typing of blood—a finding that 29 years later earned him a Nobel Prize. For years physicians had attempted to transfuse blood from one individual to another. Their ef- forts often ended in failure because the transfused blood tended to coagulate in the body of the recipient, causing instantaneous death. Landsteiner was the first to recognize that all hu- man blood was not the same; instead, he found that blood is distinguishable by its group or type. Out of Landsteiner’s work came the classification system that we call the A-B-O system. Now physicians had the key for properly matching the blood of a donor to a recipient. One blood type cannot be mixed with a different blood type without disastrous consequences. This discovery, of course, had important implications for blood transfusion and the millions of lives it has since saved. Meanwhile, Landsteiner’s findings had opened up a completely new field of research in the biological sciences. Others began to pursue the identification of ad- ditional characteristics that could further differentiate blood. By 1937, the Rh factor in blood was demonstrated, and shortly thereafter, numerous blood factors or groups were discovered. More than a hundred different blood factors have been shown to exist. However, the ones in the A-B-O system are still the most important for properly matching a donor and recipient for a transfusion.

Until the early 1990s, forensic scientists focused on blood factors, such as A-B-O, as offering the best means for linking blood to an individual. What made these factors so attrac- tive to the forensic scientist was that in theory no two individuals, except for identical twins, could be expected to have the same combination of blood factors. In other words, blood fac- tors are controlled genetically and have the potential of being a highly distinctive feature for personal identification. What makes this observation so relevant is the high frequency of oc- currence of bloodstains at crime scenes, especially crimes of the most serious nature—that is, homicides, assaults, and rapes. Consider, for example, a transfer of blood between the victim and assailant during a struggle; that is, the victim’s blood is transferred to the suspect’s gar- ment, or vice versa. If the criminalist could individualize human blood by identifying all of its known factors, the result would be evidence of the strongest kind for linking the suspect to the crime scene.

The advent of DNA technology has dramatically altered the approach of forensic scientists toward individualization of bloodstains and other biological evidence. The search for geneti- cally controlled blood factors in bloodstains has been abandoned in favor of characterizing bio- logical evidence by select regions of our deoxyribonucleic acid (DNA). The individualization of dried blood and other biological evidence, now a reality, has significantly altered the role that crime laboratories play in criminal investigations. As we will learn in the next chapter, the high sensitivity of DNA analysis has even altered the type of materials collected from crime scenes in the search for DNA. The next chapter is devoted to discussing recent breakthroughs in associating blood and semen stains with a single individual through characterization of DNA. This chapter focuses on underlying biological concepts that forensic scientists histori- cally relied on as they sought to characterize and individualize biological evidence before the dawning of the age of DNA.

The Nature of Blood The word blood actually refers to a highly complex mixture of cells, enzymes, proteins, and inorganic substances. The fluid portion of blood is called plasma. Plasma is composed princi- pally of water and accounts for 55 percent of blood content. Suspended in the plasma are solid materials consisting chiefly of cells—that is, red blood cells (erythrocytes), white blood cells (leukocytes), and platelets. The solid portion of blood accounts for 45 percent of its content. Blood clots when a protein in the plasma known as fibrin traps and enmeshes the red blood cells. If one were to remove the clotted material, a pale yellowish liquid known as serum would be left.

Obviously, considering the complexity of blood, any discussion of its function and chem- istry would have to be extensive, extending beyond the scope of this text. It is certainly far more relevant at this point to concentrate our discussion on the blood components that are

deoxyribonucleic acid (DNA) The molecules carrying the body’s genetic information; DNA is double stranded in the shape of a double helix.

plasma The fluid portion of unclotted blood.

erythrocyte A red blood cell.

serum The liquid that separates from the blood when a clot is formed.

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directly pertinent to the forensic aspects of blood identification—the red blood cells and the blood serum.

Antigens and Antibodies Functionally, red blood cells transport oxygen from the lungs to the body tissues and in turn re- move carbon dioxide from tissues by transporting it back to the lungs, where it is exhaled. How- ever, for reasons unrelated to the red blood cell’s transporting mission, on the surface of each cell are millions of characteristic chemical structures called antigens. Antigens impart blood-type characteristics to the red blood cells. Blood antigens are grouped into systems depending on their relationship to one another. More than 15 blood antigen systems have been identified to date; of these, the A-B-O and Rh systems are the most important.

If an individual is type A, this simply indicates that each red blood cell has A antigens on its surface; similarly, all type B individuals have B antigens; and the red blood cells of type AB contain both A and B antigens. Type O individuals have neither A nor B antigens on their cells. Hence, the presence or absence of the A and B antigens on the red blood cells determines a per- son’s blood type in the A-B-O system.

Another important blood antigen has been designated as the Rh factor, or D antigen. People with the D antigen are said to be Rh positive; those without this antigen are Rh negative. In routine blood banking, the presence or absence of the three antigens—A, B, and D—must be determined in testing for the compatibility of the donor and recipient.

Serum is important because it contains certain proteins known as antibodies. The fundamental principle of blood typing is that for every antigen, there exists a specific antibody. Each antibody symbol contains the prefix anti-, followed by the name of the antigen for which it is specific. Hence, anti-A is specific only for A antigen, anti-B for B antigen, and anti-D for D antigen. The serum- containing antibody is referred to as the antiserum, meaning a serum that reacts against something (antigens).

An antibody reacts only with its specific antigen, and no other. Thus, if serum containing anti-B is added to red blood cells carrying the B antigen, the two immediately combine, causing the antibody to attach itself to the cell. Antibodies are normally bivalent—that is, they have two reactive sites. This means that each antibody can simultaneously be attached to antigens located on two different red blood cells. This creates a vast network of cross-linked cells usually seen as clumping or agglutination (see Figure 14–1).

antigen A substance, usually a protein, that stimulates the body to produce antibodies against it.

antibody A protein that destroys or inacti- vates a specific antigen; antibodies are found in the blood serum.

antiserum Blood serum that contains specific antibodies.

agglutination The clumping together of red blood cells by the action of an antibody.

BA A

Anti-B

Red blood cells containing A antigens do not combine with B antibodies

B B

Anti-B

Red blood cells containing B antigens are agglutinated or clumped together in the presence of B antibodies

FIGURE 14–1 Agglutination of blood cells.

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356 CHAPTER 14

(a) (b)

FIGURE 14–2 (a) Microscopic view of normal red blood cells (500×). (b) Microscopic view of agglutinated red blood cells (500×).

serology The study of antigen–antibody reactions.

Let’s look a little more closely at this phenomenon. In normal blood, shown in Figure 14–2(a), antigens on red blood cells and antibodies coexist without destroying each other because the an- tibodies present are not specific toward any of the antigens. However, suppose a foreign serum added to the blood introduces a new antibody. The occurrence of a specific antigen–antibody reaction immediately causes the red blood cells to link together, or agglutinate, as shown in Figure 14–2(b).

Evidently, nature has taken this situation into account because when we examine the serum of type A blood, we find anti-B and no anti-A. Similarly, type B blood contains only anti-A, type O blood has both anti-A and anti-B, and type AB blood contains neither anti-A nor anti-B. The antigen and antibody components of normal blood are summarized in the fol- lowing table:

Blood Type Antigens on Red Blood Cells Antibodies in Serum

A A Anti-B

B B Anti-A

AB AB Neither anti-A nor anti-B

O Neither A nor B Both anti-A and anti-B

The reasons for the fatal consequences of mixing incompatible blood during a transfusion should now be quite obvious. For example, transfusing type A blood into a type B patient will cause the natural anti-A in the blood of the type B patient to react promptly with the incoming A antigens, resulting in agglutination. In addition, the incoming anti-B of the donor will react with the B antigens of the patient.

Blood Typing The term serology is used to describe a broad scope of laboratory tests that use specific anti- gen and serum antibody reactions. The most widespread application of serology is the typing of whole blood for its A-B-O identity. In determining the A-B-O blood type, only two antise- rums are needed—anti-A and anti-B. For routine blood typing, both of these antiserums are commercially available.

Table 14–1 summarizes how the identity of each of the four blood groups is established when the blood is tested with anti-A and anti-B serum. Type A blood is agglutinated by anti-A serum; type B blood is agglutinated by anti-B serum; type AB blood is agglutinated by both

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anti-A and anti-B; and type O blood is not agglutinated by either the anti-A or anti-B serum (see Figure 14–3).

The identification of natural antibodies present in blood offers another route to the deter- mination of blood type. Testing blood for the presence of anti-A and anti-B requires using red blood cells that have known antigens. Again, these cells are commercially available. Hence, when A cells are added to a blood specimen, agglutination occurs only in the presence of anti-A. Simi- larly, B cells agglutinate only in the presence of anti-B. All four A-B-O types can be identified in this manner by testing blood with known A and B cells, as summarized in Table 14–2.

TABLE 14–1 Identification of Blood with Known Antiserum

Anti-A Serum +

Whole Blood

Anti-B Serum +

Whole Blood Antigen Present Blood Type

+ – A A – + B B

+ + A and B AB – – Neither A nor B O

Note: + shows agglutination; – shows absence of agglutination.

TABLE 14–2 Identification of Blood with Known Cells

A Cells + Blood B Cells + Blood Antibody Present Blood Type

+ – Anti-A B – + Anti-B A

+ + Both anti-A and anti-B O – – Neither anti-A nor anti-B AB

Note: + shows agglutination; – shows absence of agglutination.

FIGURE 14–3 A blood test for types A, B, AB, and O. Commercial antisera are systematically applied to a questioned blood in order to determine blood type.

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358 CHAPTER 14

The population distribution of blood types varies with location and race throughout the world. In the United States, a typical distribution is as follows:

O A B AB

43% 42% 12% 3%

Immunoassay Techniques The concept of a specific antigen–antibody reaction is finding application in other areas unre- lated to the blood typing of individuals. Most significantly, this approach has been extended to the detection of drugs in blood and urine. Antibodies that react with drugs do not naturally exist; however, they can be produced in animals such as rabbits by first combining the drug with a protein and injecting this combination into the animal. This drug–protein complex acts as an antigen stimulating the animal to produce antibodies (see Figure 14–4). The recovered blood serum of the animal will contain antibodies that are specific or nearly specific to the drug.

Currently, thousands of individuals regularly submit to urinalysis tests for the presence of drugs of abuse. These individuals include military personnel, transportation industry employees, police and corrections personnel, and subjects requiring preemployment drug screening. Immunoassay testing for drugs has proven quite suitable for handling the large volume of specimens that must be rapidly analyzed for drug content on a daily basis. Testing laboratories have access to many commercially prepared sera arising from animals being injected with any one of a variety of drugs. A particular serum that has been added to a urine specimen is designed to interact with opiates, cannabinoids, cocaine, amphetamines, phencyclidine, barbiturates, methadone, or other drugs. A word of caution: Immunoassay is only presumptive in nature, and its result must be confirmed by additional testing. Specifically, the confirmation test of choice is gas chromatography-mass spectrometry, which is described in more detail in Chapter 11.

Forensic Characterization of Bloodstains The criminalist must answer the following questions when examining dried blood: (1) Is it blood? (2) From what species did the blood originate? (3) If the blood is of human origin, how closely can it be associated with a particular individual?

Color Tests The determination of blood is best made by means of a preliminary color test. For many years, the most commonly used test for this purpose was the benzidine color test; however, because benzidine has been identified as a known carcinogen, its use has generally been discontinued, and the chemical phenolphthalein is usually substituted in its place (this test is also known as the Kastle-Meyer color test).1 Both the benzidine and Kastle-Meyer color tests are based on the observation that blood hemoglobin possesses peroxidase-like activity. Peroxidases are

NCH3 O

Drug Protein carrier Drug antibodies

FIGURE 14–4 Stimulating production of drug antibodies

1 S. Tobe et al., “Evaluation of Six Presumptive Tests for Blood, Their Specificity, Sensitivity, and Effect on High Molecular-Weight DNA,” Journal of Forensic Sciences 52 (2007): 102–109.

hemoglobin A red blood cell protein that trans- ports oxygen in the bloodstream; it is responsible for the red color of blood.

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The quantity of chemically labeled methadone left uncombined is then measured, and this value is re- lated to the concentration of methadone originally present in the urine.

One of the most frequent uses of EMIT in foren- sic laboratories has been for screening the urine of suspected marijuana users. The primary pharmaco- logically active agent in marijuana is tetrahydrocan- nabinol, or THC. To facilitate the elimination of THC, the body converts it to a series of substances called metabolites that are more readily excreted. The major THC metabolite found in urine is a substance called THC-9-carboxylic acid. Antibodies against this me- tabolite are prepared for EMIT testing. Normally the urine of marijuana users contains a very small quantity of THC-9-carboxylic acid (less than one-millionth of a gram); however, this level is readily detected by EMIT.

The greatest problem with detecting marijuana in urine is interpretation. Although smoking marijuana will result in the detection of THC metabolite, it is dif- ficult to determine when the individual actually used marijuana. In individuals who smoke marijuana fre- quently, detection is possible within 2 to 5 days after the last use of the drug. However, some individuals may yield positive results up to 30 days after the last use of marijuana.

The Enzyme Multiplied Immunoassay Technique

Several immunological assay techniques are com- mercially available for detecting drugs through an antigen–antibody reaction. One such technique, the enzyme-multiplied immunoassay technique (EMIT), has gained widespread popularity among toxicolo- gists because of its speed and high sensitivity for de- tecting drugs in urine.

A typical EMIT analysis begins by adding to a subject’s urine antibodies that bind to a particular type or class of drug being looked for. This is followed by adding to the urine a chemically labeled version of the drug. As shown in the figure, a competition will ensue between the labeled and unlabeled drug (if it’s present in the subject’s urine) to bind with the antibody. If this competition does occur in a person’s urine, it signifies that the urine screen test was posi- tive for the drug being tested. For example, to check someone’s urine for methadone, the analyst would add methadone antibodies and chemically labeled methadone to the urine. Any methadone present in the urine immediately competes with the labeled methadone to bind with the methadone antibodies.

inside the science

1 Labeled drug

Antibody

Drug present

in urine

No drug

present in urine

* * * *

* * *

In the EMIT assay, a drug that may be present in a urine specimen will compete with added labeled drugs for a limited number of antibody binding sites. The labeled drugs are indicated by an asterisk. Once the competition for antibody sites is completed, the number of remaining unbound labeled drug is proportional to the drug’s concentration in urine.

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the process of producing antibodies designed to re- spond to foreign antigens is complex. For one, an an- tigen typically has structurally different sites to which an antibody may bind. So when the animal is actively producing attack antibodies, it produces a series of different antibodies, all of which are designed to at- tack some particular site on the antigen of interest. These antibodies are known as polyclonal antibodies.

Polyclonal and Monoclonal Antibodies

As we have seen in the previous section, when an ani- mal such as a rabbit or mouse is injected with an an- tigen, the animal responds by producing antibodies designed to bind to the invading antigen. However,

inside the science

Antigen

Antibodies Spleen cells

Malignant blood cells

Spleen cell

Hybridoma cells

Monoclonal antibodies

1. Inject mouse or rabbit with antigen.

2. Remove spleen and isolate spleen cells, which produce antibodies to the antigen of interest.

3. Fuse spleen cells with malignant cells, which grow well in culture.

4. Grow hybrid cells and isolate ones that produce the antibody of interest.

5. Culture the hybrid cells to create a virtually limitless supply of antibodies.

Steps required to produce monoclonal antibodies.

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FORENSIC SEROLOGY 361

enzymes that accelerate the oxidation of several classes of organic compounds by peroxides. When a bloodstain, phenolphthalein reagent, and hydrogen peroxide are mixed together, the blood’s hemoglobin causes the formation of a deep pink color.

The Kastle-Meyer test is not a specific test for blood; some vegetable materials, for instance, may turn Kastle-Meyer pink. These substances include potatoes and horseradish. However, it is unlikely that such materials will be encountered in criminal situations, and thus from a practical point of view, a positive Kastle-Meyer test is highly indicative of blood. Field investigators have found Hemastix strips a useful presumptive field test for blood. Designed as a urine dipstick test for blood, the strip can be moistened with distilled water and placed in contact with a suspect bloodstain. The appearance of a green color is indicative of blood.

Luminol and Bluestar Another important presumptive identification test for blood is the luminol test.2 Unlike the benzidine and Kastle-Meyer tests, the reaction of luminol with blood produces light rather than color. By spraying luminol reagent onto a suspect item, investigators can quickly screen large areas for bloodstains. The sprayed objects must be located in a darkened area while being viewed for the emission of light (luminescence); any bloodstains produce a faint blue glow. A relatively new product, Bluestar, is now available to be used in place of luminol (http://www .bluestar-forensic.com). Bluestar is easy to mix in the field. Its reaction with blood can be ob- served readily without having to create complete darkness. The luminol and Bluestar tests are extremely sensitive—capable of detecting bloodstains diluted to as little as 1 in 100,000. For this reason, spraying large areas such as carpets, walls, flooring, or the interior of a vehicle may reveal blood traces or patterns that would have gone unnoticed under normal lighting condi- tions (see Figure 14–5). It is important to note that luminol and Bluestar do not interfere with any subsequent DNA testing.3

polyclonal antibodies Antibodies produced by injecting animals with a specific antigen; a series of antibodies is produced responding to a variety of different sites on the antigen.

monoclonal antibodies A collection of identical antibodies that interact with a single antigen site.

hybridoma cells Fused spleen and tumor cells; used to produce identical monoclonal antibodies in a limitless supply.

hybridoma cells that bear the antibody activity of in- terest are then selected and cultured. The rapidly mul- tiplying cancer cells linked to the selected antibody cells produce identical monoclonal antibodies in a limitless supply, as shown in the figure.

Monoclonal antibodies are being incorporated into commercial forensic test kits with increasing frequency. Many immunoassay test kits for drugs of abuse are being formulated with monoclonal anti bodies. Also, an immunological test for seminal material that incorporates a monoclonal antibody has found wide popularity in crime laboratories (see pages 370–371).

As a side note, in 1999 the U.S. Food and Drug Administration approved a monoclonal drug treat- ment for cancer. Rituxin is a nontoxic monoclonal antibody designed to attack and destroy cancerous white blood cells containing an antigen designated as CD20. Other monoclonal drug treatments are in the pipeline. Monoclonals are finally beginning to fulfill their long-held expectation as medicine’s version of the “magic bullet.”

However, the disadvantage of polyclonal antibodies is that an animal can produce antibodies that vary in composition over time. As a result, different batches of polyclonals may vary in their specificity and their ability to bind to a particular antigen site.

As the technologies associated with forensic sci- ence have grown in importance, a need has devel- oped, in some instances, to have access to antibodies that are more uniform in their composition and attack power than the traditional polyclonals. This is best ac- complished by adopting a process in which an animal will produce antibodies designed to attack one and only one site on an antigen. Such antibodies are known as monoclonal antibodies. How can such monoclo- nals be produced? The process begins by injecting a mouse with the antigen of interest. In response, the mouse’s spleen cells will produce antibodies to fight off the invading antigen. The spleen cells are removed from the animal and are fused to fast-grow- ing blood cancer cells to produce hybridoma cells. The hybridoma cells are then allowed to multiply and are screened for their specific antibody activity. The

WEBEXTRA 14.1 See a Color Test for Blood

WEBEXTRA 14.2 See How the Hemastix Test Is Run for Blood

2 The luminol reagent is prepared by mixing 0.1 grams 3-amino-phthalhydrazide and 5.0 grams sodium carbonate in 100 milliliters distilled water. Before use, 0.7 grams sodium perborate is added to the solution.

3 A. M. Gross et al., “The Effect of Luminol on Presumptive Tests and DNA Analysis Using the Polymerase Chain Reaction,” Journal of Forensic Sciences 44 (1999): 837.

luminol The most sensitive chemical test that is capable of presumptively detecting bloodstains diluted to as little as 1 in 100,000; its reaction with blood emits light and thus requires the result to be observed in a darkened area.

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precipitin An antibody that reacts with its corresponding antigen to form a precipitate.

(a)

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FIGURE 14–5 (a) Sink and arms before applications of Bluestar reagent. (b) Bluestar applications reveals bloodstain patterns.

Microcrystalline Tests The identification of blood can be made more specific if microcrystalline tests are performed on the material. Several tests are available; the two most popular ones are the Takayama and Teichmann tests. Both of these depend on the addition of specific chemicals to the blood so that characteristic crystals with hemoglobin derivatives will form. Crystal tests are far less sensitive than color tests for blood identification and are more susceptible to interference from contami- nants that may be present in the stain.

Precipitin Test Once the stain has been characterized as blood, the serologist determines whether the stain is of human or animal origin. For this purpose, the standard test used is the precipitin test. Precipitin tests are based on the fact that when animals (usually rabbits) are injected with human blood, antibodies form that react with the invading human blood to neutralize its presence. The inves- tigator can recover these antibodies by bleeding the animal and isolating the blood serum. This serum contains antibodies that specifically react with human antigens. For this reason, the serum is known as human antiserum. In the same manner, by injecting rabbits with the blood of other known animals, virtually any kind of animal antiserum can be produced. Currently, antiserums are commercially available for humans and for a variety of commonly encountered animals—for example, dogs, cats, and deer.

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A number of techniques have been devised for performing precipitin tests on bloodstains. The classic method is to layer an extract of the bloodstain on top of the human antiserum in a capillary tube. Human blood, or for that matter, any protein of human origin in the extract, reacts specifically with antibodies present in the antiserum, as indicated by the formation of a cloudy ring or band at the interface of the two liquids (see Figure 14–6).

Gel Diffusion Another version of the precipitin test, called gel diffusion, takes advantage of the fact that an- tibodies and antigens diffuse or move toward one another on a plate coated with a gel medium made from a natural polymer called agar. The extracted bloodstain and the human antiserum are placed in separate holes opposite each other on the gel. If the blood is of human origin, a line of precipitation will form where the antigens and antibodies meet.

Similarly, the antigens and antibodies can be induced to move toward one another under the influence of an electrical field. In the electrophoretic method, an electrical potential is applied to the gel medium; a specific antigen–antibody reaction is denoted by a line of precipitation formed between the hole containing the blood extract and the hole containing the human antiserum (see Figure 14–7).

The precipitin test is very sensitive and requires only a small amount of blood for test- ing. Human bloodstains dried for 10 to 15 years and longer may still give a positive pre- cipitin reaction. Even extracts of tissue from mummies four to five thousand years old have given positive reactions with this test. Furthermore, human bloodstains diluted by washing in water and left with only a faint color may still yield a positive precipitin reaction (see Figure 14–8).

Once it has been determined that the bloodstain is of human origin, an effort must be made to associate or disassociate the stain with a particular individual. Until the mid-1990s, routine characterization of bloodstains included the determination of A-B-O types; however, the widespread use of DNA profiling or typing has relegated this subject to one of historical interest only.

Human blood

Rabbit serum

Human blood gives a precipitin band with sensitized rabbit serum

Withdrawing blood from human vein

Blood injected into rabbit

Rabbit serum sensitized to human blood is removed from rabbit

FIGURE 14–6 The precipitin test.

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FIGURE 14–8 Results of the precipitin test of dilutions of human serum up to 1 in 4,096 against a human antiserum. A reaction is visible for blood dilutions up to 1 in 256.

WEBEXTRA 14.3 Learn about the Chromosomes Present in Our Cells

gene A unit of inheritance consisting of a DNA segment located on a chromosome.

chromosome A rodlike structure in the cell nucleus, along which the genes are located; it is composed of DNA surrounded by other material, mainly proteins.

egg The female reproductive cell.

sperm The male reproductive cell.

zygote The cell arising from the union of an egg and a sperm cell.

X chromosome The female sex chromosome.

Y chromosome The male sex chromosome.

+ −

Antigen and antibody are added to their respective wells

Antigen and antibody move toward each other

Antigen and antibody have formed a visible precipitin line in the

gel between the wells

FIGURE 14–7 Antigens and antibodies moving toward one another under the influence of an electrical potential.

Principles of Heredity All of the antigens that have been described in previous sections are genetically controlled traits. That is, they are inherited from parents and become a permanent feature of a person’s biological makeup from the moment he or she is conceived. Determining the identity of these traits, then, not only provides us with a picture of how one individual compares to or differs from another, but gives us an insight into the basic biological substances that determine our overall makeup as human beings and the mechanism by which those substances are transmitted from one generation to the next.

Genes and Chromosomes Hereditary material is transmitted via microscopic units called genes. The gene is the basic unit of heredity. Each gene by itself or in concert with other genes controls the development of a spe- cific characteristic in the new individual; the genes determine the nature and growth of virtually every body structure.

The genes are positioned on chromosomes, threadlike bodies that appear in the nucleus of every body cell (see Figure 14–9). All nucleated human cells contain 46 chromosomes, mated in 23 pairs. The only exceptions are the nucleated human reproductive cells, the egg and sperm, which contain only 23 unmated chromosomes. During fertilization, a sperm and egg combine so that each contributes chromosomes to form the new cell (zygote). Hence, the new individual begins life properly with 23 mated chromosome pairs. Because the genes are positioned on the chromosomes, the new individual inherits genetic material from each parent.

Actually, two dissimilar chromosomes are involved in the determination of sex. The egg cell always contains a long chromosome known as the X chromosome, but the sperm cell may contain either a short chromosome, known as the Y chromosome, or a long X chromosome. When an X-carrying sperm fertilizes an egg, the new cell is XX and develops into a female.

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A Y-carrying sperm produces an XY fertilized egg and develops into a male. Because the sperm cell ultimately determines the nature of the chromosome pair, we can say that the father biologi- cally determines the sex of the child.

ALLELES Just as chromosomes come together in pairs, so do the genes they bear. The position a gene occupies on a chromosome is its locus. Genes that govern a given characteristic are similarly positioned on the chromosomes inherited from the mother and father. Thus, a gene for eye color on the mother’s chromosome will be aligned with a gene for eye color on the corresponding chromosome inherited from the father. Alternative forms of genes that influence a given characteristic and are aligned with one another on a chromosome pair are known as alleles.

Another simple example of allele genes in humans is that of blood types belonging to the A-B-O system. Inheritance of the A-B-O type is best described by a theory that uses three genes designated A, B, and O. A gene pair made up of two similar genes—for example, AA and BB— is said to be homozygous; a gene pair made up of two different genes—AO, for example—is said to be heterozygous. If the chromosome inherited from the father carries the A gene and the chromosome inherited from the mother carries the same gene, the offspring would have an AA combination. Similarly, if one chromosome contains the A gene and the other has the O gene, the genetic makeup of the offspring would be AO.

DOMINANT AND RECESSIVE GENES When an individual inherits two similar genes from his or her parents, there is no problem in determining the blood type of that person. Hence, an AA combination will always be type A, a BB type B, and an OO type O. However, when two different genes are inherited, one gene will be dominant. It can be said that the A and B genes are dominant and that the O gene is always recessive—that is, its characteristics remain hidden. For instance, with an AO combination, A is always dominant over O, and the individual will be typed as A. Similarly, a BO combination is typed as B. In the case of AB, the genes are codominant, and the individual’s blood type will be AB. The recessive characteristics of O appear only when both recessive genes are present. Hence, the combination OO is typed simply as O.

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locus The physical location of a gene on a chromosome.

allele Any of several alternative forms of a gene located at the same point on a particular pair of chromosomes; for example, the genes determining the blood types A and B are alleles.

FIGURE 14–9 Computer-enhanced photomicrograph image of human chromosomes.

homozygous Having two identical allelic genes on two corresponding positions of a pair of chromosomes.

heterozygous Having two different allelic genes on two corresponding positions of a pair of chromosomes.

WEBEXTRA 14.4 Learn about the Structure of Our Genes

WEBEXTRA 14.5 See How Genes Position Them- selves on a Chromosome Pair

WEBEXTRA 14.6 See How Genes Define Our Genetic Makeup

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Forensic Characterization of Semen Many cases received in a forensic laboratory involve sexual offenses, making it necessary to examine exhibits for the presence of seminal stains.

The normal male releases 2.5 to 6 milliliters of seminal fluid during an ejaculation. Each milliliter contains 100 million or more spermatozoa, the male reproductive cells. Forensic exami- nation of articles for seminal stains can actually be considered a two-step process. First, before any

Hence, in this case, 50 percent of the offspring are likely to be AO and the other 50 percent BO. These are the only genotypes possible from this combina- tion. Because O is recessive, 50 percent of the off- spring will probably be type A and 50 percent type B. From this example, we can see that no blood group gene can appear in a child unless it is present in at least one of the parents.

Although the genotyping of blood factors has useful applications for studying the transmission of blood characteristics from one generation to the next, it has no direct relevance to criminal investiga- tions. It does, however, have important implications in disputed-paternity cases, which are normally encoun- tered in civil, not criminal, courts.

Many cases of disputed paternity can be re- solved when the suspected parents and the off- spring are related according to their blood group systems. For instance, in the previous example, had the child been type AB, the suspected father would have been cleared. A type O father and a type AB mother cannot have a type AB child. On the other hand, if the child had been type A or type B, the most that could be said is that the suspect may have been the father; this does not mean that he is the father, just that he is not excluded based on blood typing. Obviously, many other males also have type O blood. Of course, the more blood group systems that are tested, the better the chances of excluding an innocent male from involvement. Conversely, if no discrepancies are found between offspring and suspect father, the more certain one can be that the suspect is indeed the father. In fact, routine pater- nity testing involves characterizing blood factors other than A-B-O. Currently, paternity testing labo- ratories have implemented tests for DNA alleles that can raise the odds of establishing paternity beyond 99 percent.

Genotypes and Phenotypes

A pair of allele genes together constitutes the genotype of the individual. However, no laboratory test can deter- mine an individual’s A-B-O genotype. For example, a person’s outward characteristic, or phenotype, may be type A, but this does not tell us whether his or her geno- type is AA or AO. The genotype can be determined only by studying the family history of the individual. If the genotypes of both parents are known, that of their possible offspring can be forecast.

An easy way to figure this out is to construct a Pun- nett square. To do this, write along a horizontal line the two genes of the male parent, and in the vertical column write the two kinds of female genes present, as shown. In our example, we assume the male parent is type O and therefore has to be an OO genotype; the female parent is type AB and can be only an AB genotype:

Father’s genotype

O O

Mother’s genotype

Next, write in each box the corresponding gene contributed from the female and then from the male. The squares will contain all the possible genotype com- binations that the parents can produce in their offspring:

O O

A AO AO

B BO BO

inside the science

genotype The particular combination of genes present in the cells of an individual.

phenotype The physical manifestation of a ge- netic trait such as shape, color, and blood type.

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tests can be conducted, the stain must be located. Considering the number and soiled condition of outer garments, undergarments, and possible bedclothing submitted for examination, this may prove to be an arduous task. Once located, the stain will have to be subjected to tests that will prove its identity; it may even be tested for the blood type of the individual from whom it originated.

Testing for Seminal Stains Often, seminal stains are readily visible on a fabric because they exhibit a stiff, crusty appear- ance. However, reliance on such appearance for locating the stain is at best unreliable and is use- ful only when the stain is present in a rather obvious area. Certainly, if the fabric has been washed or contains only minute quantities of semen, visual examination of the article offers little chance of detecting the stain. The best way to locate and characterize a seminal stain is to perform the acid phosphatase color test.

ACID PHOSPHATASE TEST Acid phosphatase is an enzyme that is secreted by the prostate gland into seminal fluid. Its concentrations in seminal fluid are up to 400 times greater than those found in any other body fluid. Its presence can easily be detected when it comes in contact with an acidic solution of sodium alpha naphthylphosphate and Fast Blue B dye. Also, 4-methyl umbelliferyl phosphate (MUP) fluoresces under UV light when it comes in contact with acid phosphatase.

The utility of the acid phosphatase test is apparent when it becomes necessary to search numerous garments or large fabric areas for seminal stains. If a filter paper is simply moistened with water and rubbed lightly over the suspect area, acid phosphatase, if present, is transferred to the filter paper. Then, when a drop or two of the sodium alpha naphthylphosphate and Fast Blue B solution are placed on the paper, the appearance of a purple color indicates the acid phosphatase enzyme. In this manner, any fabric or surface can be systematically searched for seminal stains.

If it is necessary to search extremely large areas—for example, a bedsheet or carpet—the article can be tested in sections, narrowing the location of the stain with each successive test. Alternatively, the garment under investigation can be pressed against a suitably sized piece of moistened filter paper. The paper is then sprayed with MUP solution. Semen stains appear as strongly fluorescent areas under UV light. A negative reaction can be interpreted as meaning the absence of semen. Although some vegetable and fruit juices (such as cauliflower and water- melon), fungi, contraceptive creams, and vaginal secretions give a positive response to the acid phosphatase test, none of these substances normally reacts with the speed of seminal fluid. A re- action time of less than 30 seconds is considered a strong indication of the presence of semen.

MICROSCOPIC EXAMINATION OF SEMEN Semen can be unequivocally identified by the presence of spermatozoa. When spermatozoa are located through a microscope examination, the stain is definitely identified as having been derived from semen. Spermatozoa are slender, elongated structures 50–70 microns long, each with a head and a thin flagellate tail (see Figure 14–10). The criminalist can normally locate them by immersing the stained material in a small volume of water. Rapid stirring of the liquid transfers a small percentage of the spermatozoa present into the water. A drop of the water is dried onto a microscope slide, then stained and examined under a compound microscope at a magnification of approximately 400×.4

Considering the extremely large number of spermatozoa found in seminal fluid, one would think the chance of locating one would be very good; however, this is not always true. One rea- son is that spermatozoa are bound tightly to cloth materials.5 Also, spermatozoa are extremely brittle when dry and easily disintegrate if the stain is washed or when the stain is rubbed against another object, as can happen frequently in the handling and packaging of this type of evidence. Furthermore, sexual crimes may involve males who have an abnormally low sperm count, a con- dition known as oligospermia, or they may involve individuals who have no spermatozoa at all in their seminal fluid (aspermia). Significantly, aspermatic individuals are increasing in numbers because of the growing popularity of vasectomies.

acid phosphatase An enzyme found in high concen- tration in semen.

WEBEXTRA 14.7 See How the Acid Phosphatase Test for Semen Is Run

oligospermia An abnormally low sperm count.

aspermia The absence of sperm; sterility in males.

4 J. P. Allery et al., “Cytological Detection of Spermatozoa: Comparison of Three Staining Methods,” Journal of Forensic Sciences 46 (2001): 349.

5 In one study, only a maximum of 4 sperm cells out of 1,000 could be extracted from a cotton patch and observed under the microscope. Edwin Jones (Ventura County Sheriff’s Department, Ventura, Calif.), personal communication.

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PROSTATE SPECIFIC ANTIGEN (PSA) Forensic analysts often must examine stains or swabs that they suspect contain semen (because of the presence of acid phosphatase) but that yield no detectable spermatozoa. How, then, can one unequivocally prove the presence of semen? The solution to this problem apparently came with the discovery in the 1970s of a protein called p30 or prostate specific antigen (PSA). At first, this protein was thought to be prostate specific and hence a unique identifier of semen. However, additional research has shown that low levels of p30 may be detectable in other human tissues. A more reasonable approach to the unequivocal identification of semen is to use a positive PSA (p30) in combination with an acid phosphatase color test with a reaction time of less than 30 seconds.6

When PSA is isolated and injected into a rabbit, it stimulates the production of polyclonal antibodies (anti-PSA). The sera collected from these immunized rabbits can then be used to test suspected semen stains. As shown in Figure 14–11, the stain extract is placed in one well of an electrophoretic plate and the anti-PSA in an opposite well. When an electric potential is applied, the antigens and antibodies move toward each other. The formation of a visible line midway be- tween the two wells shows the presence of PSA in the stain and proves that the stain was seminal in nature.

A more elegant approach to identifying PSA (p30) involves placing an extract of a questioned sample on a porous membrane in the presence of a monoclonal PSA antibody that is linked to a dye. If PSA is present in the extract, a PSA antigen–monoclonal PSA antibody complex forms. This complex then migrates along the membrane, where it interacts with a polyclonal PSA an- tibody embedded in the membrane. The antibody–antigen–antibody “sandwich” that forms will be apparent by the presence of a colored line (see Figure 14–12). This monoclonal antibody technique is about a hundred times more sensitive for detecting PSA than the one described in the previous paragraph.

Once the material under examination is proven to be semen, the next task is to attempt to associate the semen as closely as possible with a single individual. As we will learn in Chapter 15, forensic scientists can link seminal material to one individual with DNA tech- nology. Just as important is the knowledge that this technology can exonerate many of those wrongfully accused of sexual assault.

WEBEXTRA 14.8 See How the P30 Test Is Run

FIGURE 14–10 Photomicrograph of human spermatozoa (300×).

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Collection and Preservation of Rape Evidence Seminal constituents on a rape victim are important evidence that sexual intercourse has taken place, but their absence does not necessarily mean that a rape did not occur. Physical injuries such as bruises or bleeding tend to confirm that a violent assault did take place. Furthermore, the forceful physical contact between victim and assailant may result in a transfer of physical evidence—blood, semen, hairs, and fibers. The presence of such physical evidence will help forge a vital link in the chain of circumstances surrounding a sexual crime.

Collection and Handling To protect this kind of evidence, all the outer garments and undergarments from the involved parties should be carefully removed and packaged separately in paper (not plastic) bags. Place a clean bedsheet on the floor and lay a clean paper sheet over it. The victim must remove her shoes before standing on the paper. Have the person disrobe while standing on the paper in order to collect any loose foreign material falling from the clothing. Collect each piece of clothing as it is removed and place the items in separate paper bags to avoid

Semen extract and anti-p30 are

added to their respective wells

Antigen and antibody move toward each other

Formation of a visible precipitation line midway between the wells shows

the presence of p30 in the stain and proves the stain

is seminal in nature

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FIGURE 14–11 PSA testing by electrophoresis.

Blue dye

Human PSA (antigen) extracted from a suspect stain

Mobile monoclonal PSA antibody attached to a dye

Mobile antigen– antibody complex migrates toward reaction zone

PSA antibody Antibody– antigen– antibody sandwich seen as a blue line

Reaction zone

Positive test

FIGURE 14–12 An antibody–antigen–antibody sandwich or complex is seen as a colored band. This signifies the presence of PSA in the extract of a stain and positively identifies human semen.

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cross-contamination of physical evidence. Carefully fold the paper sheet so that all foreign materials are contained inside.

If it is deemed appropriate, bedding or the object on which the assault took place should be submitted to the laboratory for processing. Items suspected of containing seminal stains must be handled carefully. Folding an article through the stain may cause it to flake off, as will rubbing the stained area against the surface of the packaging material. If, under unusual circumstances, it is not possible to transport the stained article to the laboratory, the stained area should be cut out and submitted with an unstained piece as a substrate control.

In the laboratory, analysts try to link seminal material to a donor(s) using DNA typing. Because an individual may transfer his or her DNA types to a stain through perspiration, inves- tigators must handle stained articles with care, minimizing direct personal contact. The evidence collector must wear disposable latex gloves when such evidence must be touched.

The rape victim must undergo a medical examination as soon as possible after the assault. At this time, the appropriate items of physical evidence are collected by trained personnel. Evidence collectors should have an evidence-collection kit from the local crime laboratory (see Figures 14–13, 14–14, and 14–15).

The following items of physical evidence are to be collected:

1. Pubic combings. Place a paper towel under the buttocks and comb the pubic area for loose or foreign hairs.

2. Pubic hair standard/reference samples. Cut 15 to 20 full-length hairs from the pubic area at the skin line.

3. External genital dry-skin areas. Swab with at least one dry swab and one moistened swab. 4. Vaginal swabs and smear. Using two swabs simultaneously, carefully swab the vaginal area

and let the swabs air-dry before packaging. Using two additional swabs, repeat the swabbing procedure and smear the swabs onto separate microscope slides, allowing them to air-dry before packaging.

5. Cervix swabs. Using two swabs simultaneously, carefully swab the cervix area and let the swabs air-dry before packaging.

6. Rectal swabs and smear. To be taken when warranted by case history. Using two swabs simultaneously, swab the rectal canal, smearing one of the swabs onto a microscope slide. Allow both samples to air-dry before packaging.

7. Oral swabs and smear. To be taken if oral–genital contact occurred. Use two swabs simul- taneously to swab the buccal area and gum line. Using both swabs, prepare one smear slide. Allow both swabs and the smear to air-dry before packaging.

8. Swabs of body areas, such as breasts, suspected of being in contact with DNA arising from touching or saliva.

FIGURE 14–13 Victim rape collection kit with instructions, and forms for medical history and assault information. Envelope for foreign materials. Collection bags for outer clothing and underpants. Envelopes for debris, pubic hair combings, Envelope for pulled pubic hair. Envelopes for vaginal swabs and rectal swabs with swap boxes and microscope slides. Envelopes for oral swabs and smear with microscope and a swab box, Envelope for known saliva sample. Known blood sample envelope. Anatomical drawings.

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FIGURE 14–14 Drug Facilitated Sexual Assault Evidence Toxico logy Kit containing a blood tube and urine specimen bottle holder.

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FIGURE 14–15 Drug Facilitated Sexual Assault Evidence Toxico logy Kit containing a urine specimen bottle.

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9. Head hairs. Cut at skin line a minimum of five full-length hairs from each of the following scalp locations: center, front, back, left side, and right side. It is recommended that a total of at least 25 hairs be cut and submitted to the laboratory.

10. Blood sample. Collect at least 20 milliliters in a vacuum tube containing the preservative EDTA. The blood sample can be used for toxicological analysis if required for a drug- facilitated sexual investigation (see pages 271–273).

11. Collect buccal swab for DNA typing. 12. Fingernail scrapings. Scrape the undersurface of the nails with a dull object over a piece of

clean paper to collect debris. Use separate paper, one for each hand. 13. All clothing. Package as described earlier. 14. Urine specimen. Collect 30 milliliters or more of urine from the victim for the purpose of

conducting a drug toxicological analysis for Rohypnol, GHB, and other substances associ- ated with drug-facilitated sexual assaults (see pages 271–273).

Often during the investigation of a sexual assault, the victim reports that a perpetrator en- gaged in biting, sucking, or licking of areas of the victim’s body. As we will learn in the next chapter, the tremendous sensitivity associated with DNA technology offers investigators the op- portunity to identify a perpetuator’s DNA types from saliva residues collected off the skin. The most efficient way to recover saliva residues from the skin is to first swab the suspect area with a rotating motion using a cotton swab moistened with distilled water. A second, dry swab is then rotated over the skin to recover the moist remains on the skin’s surface from the wet swab. The swabs are air-dried and packaged together as a single sample.IS

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If a suspect is apprehended, the following items are routinely collected:

1. All clothing and any other items believed to have been worn at the time of assault. 2. Pubic hair combings. 3. Pulled head and pubic hair standard/reference samples. 4. Penile swab within 24 hours of assault when appropriate to case history. 5. Buccal swab (see pages 396–397) for DNA-typing purposes.

The advent of DNA profiling has forced investigators to rethink what items are evidential with respect to a sexual assault. As we will learn in Chapter 15, DNA levels in the range of one- billionth of a gram are now routinely characterized in crime laboratories. In the past, scant atten- tion was paid to the underwear recovered from a male who was suspected of being involved in a sexual assault. From a practical point of view, the presence of seminal constituents on a man’s underwear had little or no investigative value. Today, the high sensitivity of DNA analysis has created new areas of investigation. Experience now tells us that it is possible to establish a link between a victim and her assailant by analyzing biological material recovered from the interior front surface of a male suspect’s underwear. This is especially important when investigations have failed to yield the presence of a suspect’s DNA on exhibits recovered from the victim.

ANALYZING SEMINAL CONSTITUENTS The persistence of seminal constituents in the vagina may become a factor when trying to ascertain the time of an alleged sexual attack. Although the presence of spermatozoa in the vaginal cavity provides evidence of intercourse, important information regarding the time of sexual activity can be obtained from the knowledge that motile or living sperm generally survive up to four to six hours in the vaginal cavity of a living person. However, a successful search for motile sperm requires a microscopic examination of a vaginal smear immediately after it is taken from the victim.

A more extensive examination of vaginal collections is later made at a forensic laboratory. Non- motile sperm may be found in a living female for up to three days after intercourse and occasion- ally up to six days later. However, intact sperm (sperm with tails) are not normally found 16 hours after intercourse but have been found as late as 72 hours after intercourse. The likelihood of finding seminal acid phosphatase in the vaginal cavity markedly decreases with time following intercourse, with little chance of identifying this substance 48 hours after intercourse.6 Hence, with the possi- bility of the prolonged persistence of both spermatozoa and acid phosphatase in the vaginal cavity after intercourse, investigators should determine when and if voluntary sexual activity last occurred before the sexual assault. This information will be useful for evaluating the significance of finding these seminal constituents in the female victim. Blood or buccal swabs for DNA analysis are to be taken from any consensual partner having sex with the victim within 72 hours before the assault.

Another significant indicator of recent sexual activity is PSA. This semen marker normally is not detected in the vaginal cavity beyond 72 hours following intercourse.6

A DNA Bonus A common mode of DNA transfer occurs when skin cells from the walls of the victim’s vagina are transferred onto the suspect during intercourse. Subsequent penile contact with the inner surface of the suspect’s underwear often leads to the recovery of the female victim’s DNA from the underwear’s inner surface. The power of DNA is aptly illustrated in a case in which the female victim of a rape had consensual sexual intercourse with a male partner prior to being assaulted by

a different male. DNA extracted from the inside front area of the suspect’s underwear revealed a female DNA profile matching that of the victim. The added bonus in this case was finding male DNA on the same underwear that matched that of the consensual partner.

Based on information contained in Gary G. Verret, “Sexual Assault Cases with No Primary Transfer of Biological Material from Suspect to Victim: Evidence of Secondary and Tertiary Transfer of Biological Material from Victim to Suspect’s Undergarments,” Proceedings of the Canadian Society of Forensic Science, Toronto, Ontario, November 2001.

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WEBEXTRA 14.9 Step into the Role of the First Responding Officer at a Sexual Assault Scene

WEBEXTRA 14.10 Assume the Duties of an Evidence-Collection Technician at a Sexual Assault Scene

6 R. Dziak et al., “Providing Evidence-Based Opinions on Time Since Intercourse (TSI) Based on Body Fluid Testing Results of Internal Samples,” Canadian Society of Forensic Science Journal 44 (2011): 59.

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chapter summary The term serology describes a broad scope of laboratory tests that use specific antigen and serum antibody reactions. An antibody reacts or agglutinates only with its specific antigen. The identity of each of the four A-B-O blood groups can be established by testing the blood with anti-A and anti-B sera. The concept of specific antigen–antibody reactions has been applied to immunoassay techniques for detecting drugs of abuse in blood and urine. When an animal is injected with an antigen, its body produces a series of different antibodies, all of which are designed to attack some particular site on the antigen of interest. This collection of antibodies is known as polyclonal antibodies. Alternately, a more uniform and spe- cific collection of antibodies designed to combine with a single antigen site can be manufactured. Such antibodies are known as monoclonals.

The criminalist must answer the following questions when examining dried blood: (1) Is it blood? (2) From what species did the blood originate? (3) If the blood is of human origin, how closely can it be associated to a particular individual? The determination of blood is best made by means of a preliminary color test. A positive result from the Kastle-Meyer color test is highly indicative of blood. Alternatively, the luminol and

Bluestar tests are used to search out trace amounts of blood lo- cated at crime scenes. The precipitin test uses antisera normally derived from rabbits that have been injected with the blood of a known animal to determine the species origin of a questioned bloodstain. Before the advent of DNA typing, bloodstains were linked to a source by A-B-O typing. This approach has now been supplanted by the newer DNA technology.

Many cases sent to a forensic laboratory involve sexual offenses, making it necessary to examine exhibits for the pres- ence of seminal stains. The best way to locate and characterize a seminal stain is to perform the acid phosphatase color test. Semen can be uniquely identified by the presence of sperma- tozoa. Also, the PSA protein in combination with the acid phosphatase color test provides an unequivocal identification of semen. Forensic scientists can link seminal material to an individual by DNA typing. The rape victim must undergo a medical examination as soon as possible after the assault. At that time clothing, hairs, and vaginal and rectal swabs can be collected for subsequent laboratory examination. If a suspect is apprehended within 24 hours of the assault, it may be pos- sible to detect the victim’s DNA on the male’s underwear or on a penile swab of the suspect.

review questions

1. Karl Landsteiner discovered that blood can be classified by its ___________.

2. True or False: No two individuals, except for identical twins, can be expected to have the same combination of blood types or antigens. ___________

3. ___________ is the fluid portion of unclotted blood.

4. The liquid that separates from the blood when a clot is formed is called the ___________.

5. ___________ transport oxygen from the lungs to the body tissues and carry carbon dioxide back to the lungs.

6. On the surface of red blood cells are chemical substances called ___________, which impart blood type character- istics to the cells.

7. Type A individuals have ___________ antigens on the surface of their red blood cells.

8. Type O individuals have (both A and B, neither A nor B) antigens on their red blood cells.

9. The presence or absence of the ___________ and ___________ antigens on the red blood cells determines a person’s blood type in the A-B-O system.

10. The D antigen is also known as the ___________ antigen.

11. Serum contains proteins known as ___________, which destroy or inactivate antigens.

12. An antibody reacts with (any, only a specific) antigen.

13. True or False: Agglutination describes the clumping to- gether of red blood cells by the action of an antibody. ___________

14. Type B blood contains ___________ antigens and anti- ___________ antibodies.

15. Type AB blood has (both anti-A and anti-B, neither anti-A nor anti-B) antigens.

16. A drug–protein complex can be injected into an animal to form specific ___________ for that drug.

17. The term ___________ describes the study of antigen– antibody reactions.

18. Type AB blood (is, is not) agglutinated by both anti-A and anti-B serum.

19. Type B red blood cells agglutinate when added to type (A, B) blood.

20. Type A red blood cells agglutinate when added to type (AB, O) blood.

21. The distribution of type A blood in the United States is approximately (42, 15) percent.

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374 CHAPTER 14

22. The distribution of type AB blood in the United States is approximately (12, 3) percent.

23. (All, Most) blood hemoglobin has peroxidase-like activity.

24. For many years, the most commonly used color test for identifying blood was the ___________ color test.

25. ___________ reagent reacts with blood, causing it to luminesce.

26. Blood can be characterized as being of human origin by the ___________ test.

27. Antigens and antibodies (can, cannot) be induced to move toward each other under the influence of an elec- trical field.

28. The basic unit of heredity is the ___________.

29. Genes are positioned on threadlike bodies called ___________.

30. All nucleated cells in the human body, except the repro- ductive cells, have ___________ pairs of chromosomes.

31. The sex of an offspring is always determined by the (mother, father).

32. Genes that influence a given characteristic and are aligned with one another on a chromosome pair are known as ___________.

33. When a pair of allelic genes is identical, the genes are said to be (homozygous, heterozygous).

34. The ___________ color test is used to locate and charac- terize seminal stains.

35. Semen is unequivocally identified by the microscopic appearance of ___________.

36. Males with a low sperm count have a condition known as (oligospermia, aspermia).

37. The protein ___________ is useful for the characteriza- tion of semen.

38. True or False: DNA may be transferred to an object through the medium of perspiration. ___________

39. True or False: Seminal constituents may remain in the vagina for up to six days after intercourse. ___________

review questions for inside the science

1. An immunological assay technique used to detect the presence of minute quantities of drugs in blood and urine is ___________.

2. Antibodies designed to interact with a specific antigen site are (monoclonal, polyclonal).

3. True or False: Hybridoma cells are used to produce an- tigens designed to attack one and only one site on an antibody. ___________

4. A (phenotype, genotype) is an observable characteristic of an individual.

5. The combination of genes present in the cells of an indi- vidual is called the ___________.

6. A gene (will, will not) appear in a child when it is pres- ent in one of the parents.

7. A type B individual may have the genotype ___________ or the genotype ___________.

8. A type AB mother and type AB father will have off- spring of what possible genotypes? ___________

9. A type AB mother and type AB father will have off- spring of what possible phenotypes? ___________

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FORENSIC SEROLOGY 375

application and critical thinking

1. Police investigating the scene of a sexual assault recover a large blanket that they believe may contain useful physical evidence. They take it to the laboratory of forensic serologist Scott Alden, asking him to test it for the presence of semen. Noticing faint pink stains on the blanket, Scott asks the investigating detective if he is aware of anything that might recently have been spilled on the blanket. The detective reports that an overturned bowl of grapes and watermelon was found at the scene, as well as a broken glass that had contained wine. After the detective departs, Scott chooses and administers what he considers the best test for analyzing the piece of evidence in his possession. Three minutes

after completion of the test, the blanket shows a positive reaction. What test did Scott choose and what was his conclusion? Explain your answer.

2. Criminalist Cathy Richards is collecting evidence from the victim of a sexual assault. She places a sheet on the floor, asks the victim to disrobe, and places the cloth- ing in a paper bag. After collecting pubic combings and pubic hair samples, she takes two vaginal swabs, which she allows to air-dry before packaging. Finally, Cathy collects blood, urine, and scalp hair samples from the victim. What mistakes, if any, did she make in collecting this evidence?

further references

Jones, E. L., Jr., “The Identification and Individualization of Semen Stains,” in R. Saferstein, ed., Forensic Science Handbook, vol. 2, 2nd ed. Upper Saddle River, N.J.: Pren- tice Hall, 2005.

Shaler, R. C., “Modern Forensic Biology,” in R. Saferstein, ed., Forensic Science Handbook, vol. 1, 2nd ed. Upper Saddle River, N.J.: Prentice Hall, 2002.

Virkler, K., and I. K. Lednev, “Analysis of Body Fluids for Forensic Purposes: From Laboratory Testing to Non- Destructive Rapid Confirmatory Identification at a Crime Scene,” Forensic Science International 188 (2009): 1.

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The Grim Sleeper

The killing spree began in 1985 in Los Angeles, California, and apparently ended in 1988. All but one of the serial killer’s eight victims were black women. Many of his victims were prostitutes with whom he would have sexual contact before strangling or shooting them. In 2002, the killing resumed. The attacker was dubbed the “Grim Sleeper” because he appeared to have taken a 14-year hiatus from his crimes. By 2007, three more women were added to his list of victims. What proved particularly frustrating to investigators was that, even though this killer left behind DNA evidence at many of his crime scenes, a search of the DNA databases proved fruitless in establishing an identification. If the killer had been convicted of criminal activities in the past, they never resulted in the collection of his DNA and its placement in the California database. Finally, in 2010, police arrested and identified Lonnie David Franklin, Jr., as the Grim Sleeper. The arrest came about through a familial DNA search, which trolls through the DNA database

looking for partial DNA matches that could be linked to a close relative in the file. One prisoner—

Franklin’s son Christopher—shared a strong familial pattern with the serial killer. Investigators used DNA collected off a discarded pizza crust eaten by

Lonnie Franklin to link his DNA to the Grim Sleeper’s victims.

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